Clinical, biochemical and molecular characterization of 12 patients with pyruvate carboxylase deficiency treated with triheptanoin.

Pyruvate carboxylase (PC) deficiency is a rare autosomal recessive mitochondrial neurometabolic disorder of energy deficit resulting in high morbidity and mortality, with limited therapeutic options. The PC homotetramer has a critical role in gluconeogenesis, anaplerosis, neurotransmitter synthesis, and lipogenesis. The main biochemical and clinical findings in PC deficiency (PCD) include lactic acidosis, ketonuria, failure to thrive, and neurological dysfunction. Use of the anaplerotic agent triheptanoin on a limited number of individuals with PCD has had mixed results. We expand on the potential utility of triheptanoin in PCD by examining the clinical, biochemical, molecular, and health-related quality-of-life (HRQoL) findings in a cohort of 12 individuals with PCD (eight with Type A and two each with Types B and C) treated with triheptanoin ranging for 6 days to about 7 years. The main endpoints were changes in blood lactate and HRQoL scores, but collection of useful data was limited to about half of subjects. An overall trend of lactate reduction with time on triheptanoin was noted, but with significant variability among subjects and only one subject reaching close to statistical significance for this endpoint. Parent reported HRQoL assessments with treatment showed mixed results, with some subjects showing no change, some improvement, and some worsening of overall scores. Subjects with buried amino acids in the pyruvate carboxyltransferase domain of PC that undergo destabilizing replacements may be more likely to respond (with lactate reduction or HRQoL improvement) to triheptanoin compared to those with replacements that disrupt tetramerization or subunit-subunit interface contacts. The reason for this difference is unclear and requires further validation. We observed significant variability but an overall trend of lactate reduction with time on triheptanoin and mixed parent reported outcome changes by HRQoL assessments for subjects with PCD on long-term triheptanoin. The mixed results noted with triheptanoin therapy in this study could be due to endpoint data limitation, variability of disease severity between subjects, limitation of the parent reported HRQoL tool, or subject genotype variability. Alternative designed trials and more study subjects with PCD will be needed to validate important observations from this work.

Novel multilocus imprinting disturbances in a child with expressive language delay and intellectual disability.

Multilocus imprinting disturbances (MLID) have been associated with up to 12% of patients with Beckwith-Wiedemann syndrome, Silver-Russell syndrome, and pseudohypoparathyroidism type 1B (PHP1B). Single-gene defects affecting components of the subcortical maternal complex (SCMC) have been reported in cases with multilocus hypomethylation defects. We present a patient with speech and language impairment with mild Angelman syndrome (AS) features who demonstrates maternal hypomethylation at 15q11.2 (SNRPN) as well as 11p15.5 (KCNQ1OT1) imprinted loci, but normal methylation at 6q24.2 (PLAGL1), 7p12.1 (GRB10), 7q32.2 (MEST), 11p15.5 (H19), 14q32.2 (MEG3), 19q13.43 (PEG3), and 20q13.32 (GNAS and GNAS-AS1). The proband also has no copy number nor sequence variants within the AS imprinting center or in UBE3A. Maternal targeted next generation sequencing did not identify any pathogenic variants in ZPF57, NLRP2, NLRP5, NLRP7, KHDC3L, PADI6, TLE6, OOEP, UHRF1 or ZAR1. The presence of very delayed, yet functional speech, behavioral difficulties, EEG abnormalities but without clinical seizures, and normocephaly are consistent with the 15q11.2 hypomethylation defect observed in this patient. To our knowledge, this is the first report of MLID in a patient with mild, likely mosaic, Angelman syndrome.

Solvent accessibility of E1α and E1ß residues with known missense mutations causing pyruvate dehydrogenase complex (PDC) deficiency: Impact on PDC-E1 structure and function.

Pyruvate dehydrogenase complex deficiency is a major cause of primary lactic acidemia resulting in high morbidity and mortality, with limited therapeutic options. PDHA1 mutations are responsible for >82% of cases. The E1 component of PDC is a symmetric dimer of heterodimers (αß/α'ß') encoded by PDHA1 and PDHB. We measured solvent accessibility surface area (SASA), utilized nearest-neighbor analysis, incorporated sequence changes using mutagenesis tool in PyMOL, and performed molecular modeling with SWISS-MODEL, to investigate the impact of residues with disease-causing missense variants (DMVs) on E1 structure and function. We reviewed 166 and 13 genetically resolved cases due to PDHA1 and PDHB, respectively, from variant databases. We expanded on 102 E1α and 13 E1ß nonduplicate DMVs. DMVs of E1α Arg112-Arg224 stretch (exons 5-7) and of E1α Arg residues constituted 40% and 39% of cases, respectively, with invariant Arg349 accounting for 22% of arginine replacements. SASA analysis showed that 86% and 84% of residues with nonduplicate DMVs of E1α and E1ß, respectively, are solvent inaccessible ("buried"). Furthermore, 30% of E1α buried residues with DMVs are deleterious through perturbation of subunit-subunit interface contact (SSIC), with 73% located in the Arg112-Arg224 stretch. E1α Arg349 represented 74% of buried E1α Arg residues involved in SSIC. Structural perturbations resulting from residue replacements in some matched neighboring pairs of amino acids on different subunits involved in SSIC at 2.9-4.0 Å interatomic distance apart, exhibit similar clinical phenotype. Collectively, this work provides insight for future target-based advanced molecular modeling studies, with implications for development of novel therapeutics for specific recurrent DMVs of E1α.

Simulations of Pathogenic E1α Variants: Allostery and Impact on Pyruvate Dehydrogenase Complex-E1 Structure and Function.

Pyruvate dehydrogenase complex (PDC) deficiency is a major cause of primary lactic acidemia resulting in high morbidity and mortality, with limited therapeutic options. The E1 component of the mitochondrial multienzyme PDC (PDC-E1) is a symmetric dimer of heterodimers (αß/α'ß') encoded by the PDHA1 and PDHB genes, with two symmetric active sites each consisting of highly conserved phosphorylation loops A and B. PDHA1 mutations are responsible for 82-88% of cases. Greater than 85% of E1α residues with disease-causing missense mutations (DMMs) are solvent-inaccessible, with ∼30% among those involved in subunit-subunit interface contact (SSIC). We performed molecular dynamics simulations of wild-type (WT) PDC-E1 and E1 variants with E1α DMMs at R349 and W185 (residues involved in SSIC), to investigate their impact on human PDC-E1 structure. We evaluated the change in E1 structure and dynamics and examined their implications on E1 function with the specific DMMs. We found that the dynamics of phosphorylation Loop A, which is crucial for E1 biological activity, changes with DMMs that are at least about 15 Å away. Because communication is essential for PDC-E1 activity (with alternating active sites), we also investigated the possible communication network within WT PDC-E1 via centrality analysis. We observed that DMMs altered/disrupted the communication network of PDC-E1. Collectively, these results indicate allosteric effect in PDC-E1, with implications for the development of novel small-molecule therapeutics for specific recurrent E1α DMMs such as replacements of R349 responsible for ∼10% of PDC deficiency due to E1α DMMs.

Enantiomer-specific pharmacokinetics of D,L-3-hydroxybutyrate: Implications for the treatment of multiple acyl-CoA dehydrogenase deficiency.

D,L-3-hydroxybutyrate (D,L-3-HB, a ketone body) treatment has been described in several inborn errors of metabolism, including multiple acyl-CoA dehydrogenase deficiency (MADD; glutaric aciduria type II). We aimed to improve the understanding of enantiomer-specific pharmacokinetics of D,L-3-HB. Using UPLC-MS/MS, we analyzed D-3-HB and L-3-HB concentrations in blood samples from three MADD patients, and blood and tissue samples from healthy rats, upon D,L-3-HB salt administration (patients: 736-1123 mg/kg/day; rats: 1579-6317 mg/kg/day of salt-free D,L-3-HB). D,L-3-HB administration caused substantially higher L-3-HB concentrations than D-3-HB. In MADD patients, both enantiomers peaked at 30 to 60 minutes, and approached baseline after 3 hours. In rats, D,L-3-HB administration significantly increased Cmax and AUC of D-3-HB in a dose-dependent manner (controls vs ascending dose groups for Cmax : 0.10 vs 0.30-0.35-0.50 mmol/L, and AUC: 14 vs 58-71-106 minutes*mmol/L), whereas for L-3-HB the increases were significant compared to controls, but not dose proportional (Cmax : 0.01 vs 1.88-1.92-1.98 mmol/L, and AUC: 1 vs 380-454-479 minutes*mmol/L). L-3-HB concentrations increased extensively in brain, heart, liver, and muscle, whereas the most profound rise in D-3-HB was observed in heart and liver. Our study provides important knowledge on the absorption and distribution upon oral D,L-3-HB. The enantiomer-specific pharmacokinetics implies differential metabolic fates of D-3-HB and L-3-HB.

Impact of Diagnosis and Therapy on Cognitive Function in Urea Cycle Disorders.

OBJECTIVE: Individuals with urea cycle disorders (UCDs) often present with intellectual and developmental disabilities. The major aim of this study was to evaluate the impact of diagnostic and therapeutic interventions on cognitive outcomes in UCDs. METHODS: This prospective, observational, multicenter study includes data from 503 individuals with UCDs who had comprehensive neurocognitive testing with a cumulative follow-up of 702 patient-years. RESULTS: The mean cognitive standard deviation score (cSDS) was lower in symptomatic than in asymptomatic (p < 0.001, t test) individuals with UCDs. Intellectual disability (intellectual quotient < 70, cSDS < -2.0) was associated with the respective subtype of UCD and early disease onset, whereas height of the initial peak plasma ammonium concentration was inversely associated with neurocognitive outcomes in mitochondrial (proximal) rather than cytosolic (distal) UCDs. In ornithine transcarbamylase and argininosuccinate synthetase 1 deficiencies, we did not find evidence that monoscavenger therapy with sodium or glycerol phenylbutyrate was superior to sodium benzoate in providing cognitive protection. Early liver transplantation appears to be beneficial for UCDs. It is noteworthy that individuals with argininosuccinate synthetase 1 and argininosuccinate lyase deficiencies identified by newborn screening had better neurocognitive outcomes than those diagnosed after the manifestation of first symptoms. INTERPRETATION: Cognitive function is related to interventional and non-interventional variables. Early detection by newborn screening and early liver transplantation appear to offer greater cognitive protection, but none of the currently used nitrogen scavengers was superior with regard to long-term neurocognitive outcome. Further confirmation could determine these variables as important clinical indicators of neuroprotection for individuals with UCDs. ANN NEUROL 2019.

Enzymatic testing sensitivity, variability and practical diagnostic algorithm for pyruvate dehydrogenase complex (PDC) deficiency.

Pyruvate dehydrogenase complex (PDC) deficiency is a major cause of primary lactic acidemia in children. Prompt and correct diagnosis of PDC deficiency and differentiating between specific vs generalized, or secondary deficiencies has important implications for clinical management and therapeutic interventions. Both genetic and enzymatic testing approaches are being used in the diagnosis of PDC deficiency. However, the diagnostic efficacy of such testing approaches for individuals affected with PDC deficiency has not been systematically investigated in this disorder. We sought to evaluate the diagnostic sensitivity and variability of the various PDC enzyme assays in females and males at the Center for Inherited Disorders of Energy Metabolism (CIDEM). CIDEM data were filtered by lactic acidosis and functional PDC deficiency in at least one cell/tissue type (blood lymphocytes, cultured fibroblasts or skeletal muscle) identifying 186 subjects (51% male and 49% female), about half were genetically resolved with 78% of those determined to have a pathogenic PDHA1 mutation. Assaying PDC in cultured fibroblasts in cases where the underlying genetic etiology is PDHA1, was highly sensitive irrespective of gender; 97% (95% confidence interval [CI]: 90%-100%) and 91% (95% CI: 82%-100%) in females and males, respectively. In contrast to the fibroblast-based testing, the lymphocyte- and muscle-based testing were not sensitive (36% [95% CI: 11%-61%, p=0.0003] and 58% [95% CI: 30%-86%, p=0.014], respectively) for identifying known PDC deficient females with pathogenic PDHA1 mutations. In males with a known PDHA1 mutation, the sensitivity of the various cell/tissue assays (75% lymphocyte, 91% fibroblast and 88% muscle) were not statistically different, and the discordance frequency due to the specific cell/tissue used for assaying PDC was 0.15±0.11. Based on this data, a practical diagnostic algorithm is proposed accounting for current molecular approaches, enzyme testing sensitivity, and variability due to gender, cell/tissue type used for testing, and successive repeat testing.

Lethal neonatal case and review of primary short-chain enoyl-CoA hydratase (SCEH) deficiency associated with secondary lymphocyte pyruvate dehydrogenase complex (PDC) deficiency.

Mutations in ECHS1 result in short-chain enoyl-CoA hydratase (SCEH) deficiency which mainly affects the catabolism of various amino acids, particularly valine. We describe a case compound heterozygous for ECHS1 mutations c.836T>C (novel) and c.8C>A identified by whole exome sequencing of proband and parents. SCEH deficiency was confirmed with very low SCEH activity in fibroblasts and nearly absent immunoreactivity of SCEH. The patient had a severe neonatal course with elevated blood and cerebrospinal fluid lactate and pyruvate concentrations, high plasma alanine and slightly low plasma cystine. 2-Methyl-2,3-dihydroxybutyric acid was markedly elevated as were metabolites of the three branched-chain α-ketoacids on urine organic acids analysis. These urine metabolites notably decreased when lactic acidosis decreased in blood. Lymphocyte pyruvate dehydrogenase complex (PDC) activity was deficient, but PDC and α-ketoglutarate dehydrogenase complex activities in cultured fibroblasts were normal. Oxidative phosphorylation analysis on intact digitonin-permeabilized fibroblasts was suggestive of slightly reduced PDC activity relative to control range in mitochondria. We reviewed 16 other cases with mutations in ECHS1 where PDC activity was also assayed in order to determine how common and generalized secondary PDC deficiency is associated with primary SCEH deficiency. For reasons that remain unexplained, we find that about half of cases with primary SCEH deficiency also exhibit secondary PDC deficiency. The patient died on day-of-life 39, prior to establishing his diagnosis, highlighting the importance of early and rapid neonatal diagnosis because of possible adverse effects of certain therapeutic interventions, such as administration of ketogenic diet, in this disorder. There is a need for better understanding of the pathogenic mechanisms and phenotypic variability in this relatively recently discovered disorder.

Succinyl-CoA synthetase (SUCLA2) deficiency in two siblings with impaired activity of other mitochondrial oxidative enzymes in skeletal muscle without mitochondrial DNA depletion.

Mutations in SUCLA2 result in succinyl-CoA ligase (ATP-forming) or succinyl-CoA synthetase (ADP-forming) (A-SCS) deficiency, a mitochondrial tricarboxylic acid cycle disorder. The phenotype associated with this gene defect is largely encephalomyopathy. We describe two siblings compound heterozygous for SUCLA2 mutations, c.985A>G (p.M329V) and c.920C>T (p.A307V), with parents confirmed as carriers of each mutation. We developed a new LC-MS/MS based enzyme assay to demonstrate the decreased SCS activity in the siblings with this unique genotype. Both siblings shared bilateral progressive hearing loss, encephalopathy, global developmental delay, generalized myopathy, and dystonia with choreoathetosis. Prior to diagnosis and because of lactic acidosis and low activity of muscle pyruvate dehydrogenase complex (PDC), sibling 1 (S1) was placed on dichloroacetate, while sibling 2 (S2) was on a ketogenic diet. S1 developed severe cyclic vomiting refractory to therapy, while S2 developed Leigh syndrome, severe GI dysmotility, intermittent anemia, hypogammaglobulinemia and eventually succumbed to his disorder. The mitochondrial DNA contents in skeletal muscle (SM) were normal in both siblings. Pyruvate dehydrogenase complex, ketoglutarate dehydrogenase complex, and several mitochondrial electron transport chain (ETC) activities were low or at the low end of the reference range in frozen SM from S1 and/or S2. In contrast, activities of PDC, other mitochondrial enzymes of pyruvate metabolism, ETC and, integrated oxidative phosphorylation, in skin fibroblasts were not significantly impaired. Although we show that propionyl-CoA inhibits PDC, it does not appear to account for decreased PDC activity in SM. A better understanding of the mechanisms of phenotypic variability and the etiology for tissue-specific secondary deficiencies of mitochondrial enzymes of oxidative metabolism, and independently mitochondrial DNA depletion (common in other cases of A-SCS deficiency), is needed given the implications for control of lactic acidosis and possible clinical management.

The M405V allele of the glutaryl-CoA dehydrogenase gene is an important marker for glutaric aciduria type I (GA-I) low excretors.

Glutaric aciduria type I (GA-I) is an autosomal recessive organic aciduria resulting from a functional deficiency of glutaryl-CoA dehydrogenase, encoded by GCDH. Two clinically indistinguishable diagnostic subgroups of GA-I are known; low and high excretors (LEs and HEs, respectively). Early medical and dietary interventions can result in significantly better outcomes and improved quality of life for patients with GA-I. We report on nine cases of GA-I LE patients all sharing the M405V allele with two cases missed by newborn screening (NBS) using tandem mass spectrometry (MS/MS). We describe a novel case with the known pathogenic M405V variant and a novel V133L variant, and present updated and previously unreported clinical, biochemical, functional and molecular data on eight other patients all sharing the M405V allele. Three of the nine patients are of African American ancestry, with two as siblings. GCDH activity was assayed in six of the nine patients and varied from 4 to 25% of the control mean. We support the use of urine glutarylcarnitine as a biochemical marker of GA-I by demonstrating that glutarylcarnitine is efficiently cleared by the kidney (50-90%) and that plasma and urine glutarylcarnitine follow a linear relationship. We report the allele frequencies for three known GA-I LE GCDH variants (M405V, V400M and R227P) and note that both the M405V and V400M variants are significantly more common in the population of African ancestry compared to the general population. This report highlights the M405V allele as another important molecular marker in patients with the GA-I LE phenotype. Therefore, the incorporation into newborn screening of molecular screening for the M405V and V400M variants in conjunction with MS/MS could help identify asymptomatic at-risk GA-I LE patients that could potentially be missed by current NBS programs.

Age-related effect of serotonin transporter genotype on amygdala and prefrontal cortex function in adolescence.

The S and LG alleles of the serotonin transporter-linked polymorphic region (5-HTTLPR) lower serotonin transporter expression. These low-expressing alleles are linked to increased risk for depression and brain activation patterns found in depression (increased amygdala activation and decreased amygdala-prefrontal cortex connectivity). Paradoxically, serotonin transporter blockade relieves depression symptoms. Rodent models suggest that decreased serotonin transporter in early life produces depression that emerges in adolescence, whereas decreased serotonin transporter that occurs later in development ameliorates depression. However, no brain imaging research has yet investigated the moderating influence of human development on the link between 5-HTTLPR and effect-related brain function. We investigated the age-related effect of 5-HTTLPR on amygdala activation and amygdala-prefrontal cortex connectivity using a well-replicated probe, an emotional face task, in children and adolescents aged 9-19 years. A significant genotype-by-age interaction predicted amygdala activation, such that the low-expressing genotype (S/S and S/LG ) group showed a greater increase in amygdala activation with age compared to the higher expressing (LA /LA and S/LA ) group. Additionally, compared to the higher expressing group, the low-expressing genotype group exhibited decreased connectivity between the right amygdala and ventromedial prefrontal cortex with age. Findings indicate that low-expressing genotypes may not result in the corticolimbic profile associated with depression risk until later adolescence.

A novel GK Ala469Val variant resulting in glycerol kinase deficiency with concurrent hepatoblastoma: A case report.

Glycerol kinase deficiency (GKD) is a rare X-linked condition where glycerol cannot be phosphorylated to glycerol-3-phosphate, a key component of gluconeogenesis. Clinical presentation varies widely. We present a novel variant of the responsible GK in a patient with concurrent hepatoblastoma, whose course was complicated by hypoglycemia. Hepatoblastoma has not previously been described with GKD, highlighting the need for further research into GKD and its potential role in the pathogenesis of some forms of hepatoblastoma.

Amino acid ratio combinations as biomarkers for discriminating patients with pyruvate dehydrogenase complex deficiency from other inborn errors of metabolism.

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.

Are asymptomatic carriers of OTC deficiency always asymptomatic? A multicentric retrospective study of risk using the UCDC longitudinal study database.

BACKGROUND: Ornithine transcarbamylase deficiency (OTCD) due to an X-linked OTC mutation, is responsible for moderate to severe hyperammonemia (HA) with substantial morbidity and mortality. About 80% of females with OTCD remain apparently "asymptomatic" with limited studies of their clinical characteristics and long-term health vulnerabilities. Multimodal neuroimaging studies and executive function testing have shown that asymptomatic females exhibit limitations when stressed to perform at higher cognitive load and had reduced activation of the prefrontal cortex. This retrospective study aims to improve understanding of factors that might predict development of defined complications and serious illness in apparent asymptomatic females. A proband and her daughter are presented to highlight the utility of multimodal neuroimaging studies and to underscore that asymptomatic females with OTCD are not always asymptomatic. METHODS: We review data from 302 heterozygote females with OTCD enrolled in the Urea Cycle Disorders Consortium (UCDC) longitudinal natural history database. We apply multiple neuroimaging modalities in the workup of a proband and her daughter. RESULTS: Among the females in the database, 143 were noted as symptomatic at baseline (Sym). We focused on females who were asymptomatic (Asx, n = 111) and those who were asymptomatic initially upon enrollment in study but who later became symptomatic sometime during follow-up (Asx/Sym, n = 22). The majority of Asx (86%) and Asx/Sym (75%) subjects did not restrict protein at baseline, and ~38% of Asx and 33% of Asx/Sym subjects suffered from mild to severe neuropsychiatric conditions such as mood disorder and sleep problems. The risk of mild to severe HA sometime later in life for the Asx and Asx/Sym subjects as a combined group was ~4% (5/133), with ammonia ranging from 77 to 470 µM and at least half (2/4) of subjects requiring hospital admission and nitrogen scavenger therapy. For this combined group, the median age of first HA crisis was 50 years, whereas the median age of first symptom which included neuropsychiatric and/or behavioral symptoms was 17 years. The multimodal neuroimaging studies in female heterozygotes with OTCD also underscore that asymptomatic female heterozygotes with OTCD (e.g., proband) are not always asymptomatic. CONCLUSIONS: Analysis of Asx and Asx/Sym females with OTCD in this study suggests that future evidence-based management guidelines and/or a clinical risk score calculator for this cohort could be useful management tools to reduce morbidity and improve long-term quality of life.

The impact of serotonin transporter (5-HTTLPR) genotype on the development of resting-state functional connectivity in children and adolescents: a preliminary report.

A fundamental component of brain development is the formation of large-scale networks across the cortex. One such network, the default network, undergoes a protracted development, displaying weak connectivity in childhood that strengthens in adolescence and becomes most robust in adulthood. Little is known about the genetic contributions to default network connectivity in adulthood or during development. Alterations in connectivity between posterior and frontal portions of the default network have been associated with several psychological disorders, including anxiety, autism spectrum disorders, schizophrenia, depression, and attention-deficit/hyperactivity disorder. These disorders have also been linked to variants of the serotonin transporter linked polymorphic region (5-HTTLPR). The LA allele of 5-HTTLPR results in higher serotonin transporter expression than the S allele or the rarer LG allele. 5-HTTLPR may influence default network connectivity, as the superior medial frontal region has been shown to be sensitive to changes in serotonin. Also, serotonin as a growth factor early in development may alter large-scale networks such as the default network. The present study examined the influence of 5-HTTLPR variants on connectivity between the posterior and frontal structures and its development in a cross-sectional study of 39 healthy children and adolescents. We found that children and adolescents homozygous for the S allele (S/S, n=10) showed weaker connectivity in the superior medial frontal cortex compared to those homozygous for the LA allele (LA/LA, n=13) or heterozygotes (S/LA, S/LG, n=16). Moreover, there was an age-by-genotype interaction, such that those with LA/LA genotype had the steepest age-related increase in connectivity between the posterior hub and superior medial frontal cortex, followed by heterozygotes. In contrast, individuals with the S/S genotype had the least age-related increase in connectivity strength. This preliminary report expands our understanding of the genetic influences on the development of large-scale brain connectivity and lays down the foundation for future research and replication of the results with a larger sample.

A complex 6p25 rearrangement in a child with multiple epiphyseal dysplasia.

Genomic rearrangements are increasingly recognized as important contributors to human disease. Here we report on an 11½-year-old child with myopia, Duane retraction syndrome, bilateral mixed hearing loss, skeletal anomalies including multiple epiphyseal dysplasia, and global developmental delay, and a complex 6p25 genomic rearrangement. We have employed oligonucleotide-based comparative genomic hybridization arrays (aCGH) of different resolutions (44 and 244K) as well as a 1 M single nucleotide polymorphism (SNP) array to analyze this complex rearrangement. Our analyses reveal a complex rearrangement involving a ∼2.21 Mb interstitial deletion, a ∼240 kb terminal deletion, and a 70-80 kb region in between these two deletions that shows maintenance of genomic copy number. The interstitial deletion contains eight known genes, including three Forkhead box containing (FOX) transcription factors (FOXQ1, FOXF2, and FOXC1). The region maintaining genomic copy number partly overlaps the dual specificity protein phosphatase 22 (DUSP22) gene. Array analyses suggest a homozygous loss of genomic material at the 5' end of DUSP22, which was corroborated using TaqMan® copy number analysis. It is possible that this homozygous genomic loss may render both copies of DUSP22 or its products non-functional. Our analysis suggests a rearrangement mechanism distinct from a previously reported replication-based error-prone mechanism without template switching for a specific 6p25 rearrangement with a 1.22 Mb interstitial deletion. Our study demonstrates the utility and limitations of using oligonucleotide-based aCGH and SNP array technologies of increasing resolutions in order to identify complex DNA rearrangements and gene disruptions.

Novel presentations associated with a PDHA1 variant - Alternating hemiplegia in Hemizygote proband and Guillain Barre Syndrome in Heterozygote mother.

We report a 5-year-old male with a PDHA1 variant who presented with alternating hemiplegia of childhood and later developed developmental regression, basal ganglia injury and episodic lactic acidosis. Enzyme assay in lymphocytes confirmed a diagnosis of Pyruvate Dehydrogenase Complex (PDC) deficiency. His mother who was heterozygous for the same variant suffered from ophthalmoplegia, chronic migraine and developed flaccid paralysis at 36 years of age. PDHA1 is the most common genetic cause of PDC deficiency and presents with a myriad of neurological phenotypes including neonatal form with lactic acidosis, non-progressive infantile encephalopathy, Leigh syndrome subtype and intermittent ataxia. The presentations in our 2 patients contribute to the clinical heterogeneity of this neurogenetic condition.

A Novel Homozygous Missense Mutation in the YARS Gene: Expanding the Phenotype of YARS Multisystem Disease.

Aminoacyl-tRNA synthetases (ARSs) are crucial enzymes for protein translation. Mutations in genes encoding ARSs are associated with human disease. Tyrosyl-tRNA synthetase is encoded by YARS which is ubiquitously expressed and implicated in an autosomal dominant form of Charcot-Marie-Tooth and autosomal recessive YARS-related multisystem disease. We report on a former 34-week gestational age male who presented at 2 months of age with failure to thrive (FTT) and cholestatic hepatitis. He was subsequently diagnosed with hyperinsulinemic hypoglycemia with a negative congenital hyperinsulinism gene panel and F-DOPA positron-emission tomography (PET) scan that did not demonstrate a focal lesion. Autopsy findings were notable for overall normal pancreatic islet size and morphology. Trio whole exome sequencing identified a novel homozygous variant of uncertain significance in YARS (c.611A > C, p.Tyr204Cys) with each parent a carrier for the YARS variant. Euglycemia was maintained with diazoxide (max dose, 18 mg/kg/day), and enteral dextrose via gastrostomy tube (G-Tube). During his prolonged hospitalization, the patient developed progressive liver disease, exocrine pancreatic insufficiency, acute renal failure, recurrent infections, ichthyosis, hematologic concerns, hypotonia, and global developmental delay. Such multisystem features have been previously reported in association with pathogenic YARS mutations. Although hypoglycemia has been associated with pathogenic YARS mutations, this report provides more conclusive data that a YARS variant can cause hyperinsulinemic hypoglycemia. This case expands the allelic and clinical heterogeneity of YARS-related disease. In addition, YARS-related disease should be considered in the differential of hyperinsulinemic hypoglycemia associated with multisystem disease.

Duplication 16p11.2 in a child with infantile seizure disorder.

Submicroscopic recurrent 16p11.2 rearrangements are associated with several neurodevelopmental disorders, including autism, mental retardation, and schizophrenia. The common 16p11.2 region includes 24 known genes, of which 22 are expressed in the developing human fetal nervous system. As yet, the mechanisms leading to neurodevelopmental abnormalities and the broader phenotypes associated with deletion or duplication of 16p11.2 have not been clarified. Here we report a child with spastic quadriparesis, refractory infantile seizures, severe global developmental delay, hypotonia, and microcephaly, and a de novo 598 kb 16p11.2 microduplication. Family history is negative for any of these features in parents and immediate family members. Sequencing analyses showed no mutations in DOC2A, QPRT, and SEZ6L2, genes within the duplicated 16p11.2 region that have been implicated in neuronal function and/or seizure related phenotypes. The child's clinical course is consistent with a rare seizure disorder called malignant migrating partial seizure disorder of infancy, raising the possibility that duplication or disruption of genes in the 16p11.2 interval may contribute to this severe disorder.

Utility of specific amino acid ratios in screening for pyruvate dehydrogenase complex deficiencies and other mitochondrial disorders associated with congenital lactic acidosis and newborn screening prospects.

Pyruvate dehydrogenase complex deficiencies (PDCDs) and other mitochondrial disorders (MtDs) can (a) result in congenital lactic acidosis with elevations of blood alanine (Ala) and proline (Pro), (b) lead to decreased ATP production, and (c) result in high morbidity and mortality. With ~140,000 live births annually in Ohio and ~1 in 9,000 overall prevalence of MtDs, we estimate 2 to 3 newborns will have PDCD and 13 to 14 others likely will have another MtD annually. We compared the sensitivities of plasma amino acids (AA) Alanine (Ala), Alanine:Leucine (Ala:Leu), Alanine:Lysine and the combination of Ala:Leu and Proline:Leucine (Pro:Leu), in subjects with known primary-specific PDCD due to PDHA1 and PDHB mutations vs controls. Furthermore, in collaboration with the Ohio newborn screening (NBS) laboratory, we determined Ala and Pro concentrations in dried blood spot (DBS) specimens using existing NBS analytic approaches and evaluated Ala:Leu and Pro:Leu ratios from DBS specimens of 123,414 Ohio newborns in a 12-month period. We used the combined Ala:Leu ≥4.0 and Pro:Leu ≥3.0 ratio criterion from both DBS and plasma specimens as a screening tool in our retrospective review of newborn data. The screening tool applied on DBS and/or plasma (or serum) AA specimens successfully identified three unrelated females with novel de novo PDHA1 mutations, one male with a novel de novo X-linked HSD17B10 mutation, and a female with VARS2 mutations. This work lays the first step for piloting an NBS protocol in Ohio for identifying newborns at high risk for primary-specific PDCD and other MtDs who might benefit from neonatal diagnosis and early institution of known therapy and/or potential novel therapies for such disorders.