Hypoglycemia in a Pediatric Emergency Department: Single-Center Experience on 402 Children.

OBJECTIVES: This study aimed to establish the rate, etiology, and short-term outcome of hypoglycemia in infants and children accessing an emergency department of a tertiary care pediatric hospital. METHODS: The study was retrospectively conducted on the clinical records of children with hypoglycemia aged 15 days to 17 years who were admitted consecutively to the emergency department during a 6-year period for various clinical conditions. Hypoglycemia was defined as a venous plasma glucose level lower than 45 mg/dL. RESULTS: Hypoglycemia was detected in 402 patients (female-to-male ratio, 1.26; mean age, 2.6 ± 1.8 years), with a rate of 0.99 per 1000 children. Plasma glucose levels ranged from 3 to 45 (mean, 37.48 ± 7.44) mg/dL. Hypoglycemia was associated with gastroenteritis or other infectious diseases causing protracted fasting in 86.32% of cases, whereas hypoglycemia related to a different etiology (HDE) was observed in 13.68% of hypoglycemic children. Most HDE patients had a final diagnosis of ketotic hypoglycemia, whereas metabolic defects were a rare (1.49%) but nonnegligible etiologic cause. A severe triage code was more frequent in the HDE group (P < 0.001). Factors significantly and independently associated with HDE were impaired level of consciousness, assessed with the AVPU scale (A, alert; V, responding to verbal; P, responding to pain; U, unresponsive; adjusted odds ratio, 2.50; P = 0.025) and clinical onset within 12 hours (adjusted odds ratio, 3.98; P < 0.001). CONCLUSIONS: In a nonnegligible number of critically ill children, hypoglycemia can be detected. In a minority of cases, hypoglycemia was due to metabolic disorders that should be suspected on the basis of the severity of hypoglycemia, and the recent onset and the presence of neuroglycopenic symptoms.

Combined proteomic and lipidomic studies in Pompe disease allow a better disease mechanism understanding.

Pompe disease (PD) is caused by deficiency of the enzyme acid α-glucosidase resulting in glycogen accumulation in lysosomes. Clinical symptoms include skeletal myopathy, respiratory failure, and cardiac hypertrophy. We studied plasma proteomic and lipidomic profiles in 12 PD patients compared to age-matched controls. The proteomic profiles were analyzed by nLC-MS/MS SWATH method. Wide-targeted lipidomic analysis was performed by LC-IMS/MS, allowing to quantify >1100 lipid species, spanning 13 classes. Significant differences were found for 16 proteins, with four showing the most relevant changes (GPLD1, PON1, LDHB, PKM). Lipidomic analysis showed elevated levels of three phosphatidylcholines and of the free fatty acid 22:4, and reduced levels of six lysophosphatidylcholines. Up-regulated glycolytic enzymes (LDHB and PKM) are involved in autophagy and glycogen metabolism, while down-regulated PON1 and GPLD1 combined with lipidomic data indicate an abnormal phospholipid metabolism. Reduced GPLD1 and dysregulation of lipids with acyl-chains characteristic of GPI-anchor structure suggest the potential involvement of GPI-anchor system in PD. Results of proteomic analysis displayed the involvement of multiple cellular functions affecting inflammatory, immune and antioxidant responses, autophagy, Ca2+ -homeostasis, and cell adhesion. The combined multi-omic approach revealed new biosignatures in PD, providing novel insights in disease pathophysiology with potential future clinical application.

Delayed appearance of 3-methylglutaconic aciduria in neonates with early onset metabolic cardiomyopathies: A potential pitfall for the diagnosis.

Infantile onset cardiomyopathies are highly heterogeneous with several phenocopies compared with adult cardiomyopathies. Multidisciplinary management is essential in determining the underlying etiology in children's cardiomyopathy. Elevated urinary excretion of 3-methylglutaconic acid (3-MGA) is a useful tool in identifying the etiology in some metabolic cardiomyopathy. Here, we report the delayed appearance of 3-MGA-uria, between 6 and 18 months in three patients (out of 100 childhood onset cardiomyopathy) with neonatal onset cardiomyopathy, secondary to TMEM70 mutations and TAZ mutations (Barth syndrome), in whom extensive metabolic investigations, performed in the first weeks of life, did not display 3-MGA-uria. Serial retrospective evaluations showed full characteristic features of TMEM70 and TAZ mutations (Barth syndrome) in these three patients, including a clearly abnormal monolysocardiolipin/cardiolipin ratio in the two Barth syndrome patients. Serially repeated metabolic investigations finally discovered the 3-MGA-uria biomarker in all three patients between the age of 6 and 18 months. Our observation provides novel insights into the temporal appearance of 3-MGA-uria in TMEM70 and TAZ mutations (Barth syndrome) and focus the importance of multidisciplinary management and careful evaluation of family history and red flag signs for phenocopies in infantile onset cardiomyopathies.

Plasma methylcitric acid and its correlations with other disease biomarkers: The impact in the follow up of patients with propionic and methylmalonic acidemia.

Methylcitric acid (MCA) analysis has been mainly utilized for the diagnosis of propionate disorders or as a second-tier test in newborn screening, but its utility for patients monitoring still needs to be established. We explored the potential contribution of MCA in the long-term management of organic acidurias. We prospectively evaluated plasma MCA and its relationship with disease biomarkers, clinical status, and disease burden in 22 patients, 13 with propionic acidemia (PA) and nine with methylmalonic acidemia (MMA) on standard treatment and/or after transplantation. Samples were collected at scheduled routine controls or during episodes of metabolic decompensation (MD), 10 patients were evaluated after transplantation (six liver, two combined liver and kidney, 2 kidney). MCA levels were higher in PA compared to MMA and its levels were not influenced by the clinical status (MD vs well state). In MMA, MCA was higher in elder patients and, along with fibroblast growth factor 21 (FGF21) and plasma methylmalonic acid, negatively correlated with GFR. In both diseases, MCA correlated with ammonia, glycine, lysine, C3, and the C3/C2, C3/C16 ratios. The disease burden showed a direct correlation with MCA and FGF21, for both diseases. All transplanted patients showed a significant reduction of MCA in comparison to baseline values, with some differences dependent on the type of transplantation. Our study provided new insights in understanding the disease pathophysiology, showing similarities between MCA and FGF21 in predicting disease burden, long-term complications and in evaluating the impact of organ transplantation.

microRNAs as biomarkers in Pompe disease.

PURPOSE: We studied microRNAs as potential biomarkers for Pompe disease. METHODS: We analyzed microRNA expression by small RNA-seq in tissues from the disease murine model at two different ages (3 and 9 months), and in plasma from Pompe patients. RESULTS: In the mouse model we found 211 microRNAs that were differentially expressed in gastrocnemii and 66 in heart, with a different pattern of expression at different ages. In a preliminary analysis in plasma from six patients 55 microRNAs were differentially expressed. Sixteen of these microRNAs were common to those dysregulated in mouse tissues. These microRNAs are known to modulate the expression of genes involved in relevant pathways for Pompe disease pathophysiology (autophagy, muscle regeneration, muscle atrophy). One of these microRNAs, miR-133a, was selected for further quantitative real-time polymerase chain reaction analysis in plasma samples from 52 patients, obtained from seven Italian and Dutch biobanks. miR-133a levels were significantly higher in Pompe disease patients than in controls and correlated with phenotype severity, with higher levels in infantile compared with late-onset patients. In three infantile patients miR-133a decreased after start of enzyme replacement therapy and evidence of clinical improvement. CONCLUSION: Circulating microRNAs may represent additional biomarkers of Pompe disease severity and of response to therapy.

Mis-splicing of the GALNS gene resulting from deep intronic mutations as a cause of Morquio a disease.

BACKGROUND: Mucopolysaccharidosis-IVA (Morquio A disease) is a lysosomal disorder in which the abnormal accumulation of keratan sulfate and chondroitin-6-sulfate is consequent to mutations in the galactosamine-6-sulfatase (GALNS) gene. Since standard DNA sequencing analysis fails to detect about 16% of GALNS mutant alleles, gross DNA rearrangement screening and uniparental disomy evaluation are required to complete the molecular diagnosis. Despite this, the second pathogenic GALNS allele generally remains unidentified in ~ 5% of Morquio-A disease patients. METHODS: In an attempt to bridge the residual gap between clinical and molecular diagnosis, we performed an mRNA-based evaluation of three Morquio-A disease patients in whom the second mutant GALNS allele had not been identified. We also performed sequence analysis of the entire GALNS gene in two patients. RESULTS: Different aberrant GALNS mRNA transcripts were characterized in each patient. Analysis of these transcripts then allowed the identification, in one patient, of a disease-causing deep intronic GALNS mutation. The aberrant mRNA products identified in the other two individuals resulted in partial exon loss. Despite sequencing the entire GALNS gene region in these patients, the identity of a single underlying pathological lesion could not be unequivocally determined. We postulate that a combination of multiple variants, acting in cis, may synergise in terms of their impact on the splicing machinery. CONCLUSIONS: We have identified GALNS variants located within deep intronic regions that have the potential to impact splicing. These findings have prompted us to incorporate mRNA analysis into our diagnostic flow procedure for the molecular analysis of Morquio A disease.

Persistent Hypoglycemia in Children: Targeted Gene Panel Improves the Diagnosis of Hypoglycemia Due to Inborn Errors of Metabolism.

OBJECTIVES: To evaluate the role of next generation sequencing in genetic diagnosis of pediatric patients with persistent hypoglycemia. STUDY DESIGN: Sixty-four patients investigated through an extensive workup were divided in 3 diagnostic classes based on the likelihood of a genetic diagnosis: (1) single candidate gene (9/64); (2) multiple candidate genes (43/64); and (3) no candidate gene (12/64). Subsequently, patients were tested through a custom gene panel of 65 targeted genes, which included 5 disease categories: (1) hyperinsulinemic hypoglycemia, (2) fatty acid-oxidation defects and ketogenesis defects, (3) ketolysis defects, (4) glycogen storage diseases and other disorders of carbohydrate metabolism, and (5) mitochondrial disorders. Molecular data were compared with clinical and biochemical data. RESULTS: A proven diagnosis was obtained in 78% of patients with suspicion for a single candidate gene, in 49% with multiple candidate genes, and in 33% with no candidate gene. The diagnostic yield was 48% for hyperinsulinemic hypoglycemia, 66% per fatty acid-oxidation and ketogenesis defects, 59% for glycogen storage diseases and other carbohydrate disorders, and 67% for mitochondrial disorders. CONCLUSIONS: This approach provided a diagnosis in ~50% of patients in whom clinical and laboratory evaluation did not allow identification of a single candidate gene and a diagnosis was established in 33% of patients belonging to the no candidate gene class. Next generation sequencing technique is cost-effective compared with Sanger sequencing of multiple genes and represents a powerful tool for the diagnosis of inborn errors of metabolism presenting with persistent hypoglycemia.

Muscle MRI of classic infantile pompe patients: Fatty substitution and edema-like changes.

INTRODUCTION: The aim of this study was to evaluate the muscle MRI pattern of 9 patients (median age: 6.5 ± 2.74 years) affected by classic infantile-onset Pompe disease who were treated with enzyme replacement therapy. METHODS: We performed and qualitatively scored T1-weighted (T1-w) sequences of the facial, shoulder girdle, paravertebral, and lower limb muscles and short-tau inversion recovery (STIR) sequences of the lower limbs using the Mercuri and Morrow scales, respectively. RESULTS: On T1-w images, mild (grade 1) or moderate (grade 2) involvement was found in the tongue in 6 of 6 patients and in the adductor magnus muscle in 6 of 9. STIR hyperintensity was detected in all areas examined and was categorized as limited to mild in 5 of 8 patients. CONCLUSIONS: On T1-w sequences, mild/moderate adipose substitution in the adductor magnus and tongue muscles was documented. STIR edema-like alterations of thigh and calf muscles are novel findings. Correlations with biopsy findings and clinical parameters are needed to fully understand these findings. Muscle Nerve 55: 841-848, 2017.

The treatment of juvenile/adult GM1-gangliosidosis with Miglustat may reverse disease progression.

Juvenile and adult GM1-gangliosidosis are invariably characterized by progressive neurological deterioration. To date only symptomatic therapies are available. We report for the first time the positive results of Miglustat (OGT 918, N-butyl-deoxynojirimycin) treatment on three Italian GM1-gangliosidosis patients. The first two patients had a juvenile form (enzyme activity ≤5%, GLB1 genotype p.R201H/c.1068 + 1G > T; p.R201H/p.I51N), while the third patient had an adult form (enzyme activity about 7%, p.T329A/p.R442Q). Treatment with Miglustat at the dose of 600 mg/day was started at the age of 10, 17 and 28 years; age at last evaluation was 21, 20 and 38 respectively. Response to treatment was evaluated using neurological examinations in all three patients every 4-6 months, the assessment of Movement Disorder-Childhood Rating Scale (MD-CRS) in the second patient, and the 6-Minute Walking Test (6-MWT) in the third patient. The baseline neurological status was severely impaired, with loss of autonomous ambulation and speech in the first two patients, and gait and language difficulties in the third patient. All three patients showed gradual improvement while being treated; both juvenile patients regained the ability to walk without assistance for few meters, and increased alertness and vocalization. The MD-CRS class score in the second patient decreased from 4 to 2. The third patient improved in movement and speech control, the distance covered during the 6-MWT increased from 338 to 475 m. These results suggest that Miglustat may help slow down or reverse the disease progression in juvenile/adult GM1-gangliosidosis.

Evaluation of plasma cholestane-3ß,5α,6ß-triol and 7-ketocholesterol in inherited disorders related to cholesterol metabolism.

Oxysterols are intermediates of cholesterol metabolism and are generated from cholesterol via either enzymatic or nonenzymatic pathways under oxidative stress conditions. Cholestan-3ß,5α,6ß-triol (C-triol) and 7-ketocholesterol (7-KC) have been proposed as new biomarkers for the diagnosis of Niemann-Pick type C (NP-C) disease, representing an alternative tool to the invasive and time-consuming method of fibroblast filipin test. To test the efficacy of plasma oxysterol determination for the diagnosis of NP-C, we systematically screened oxysterol levels in patients affected by different inherited disorders related with cholesterol metabolism, which included Niemann-Pick type B (NP-B) disease, lysosomal acid lipase (LAL) deficiency, Smith-Lemli-Opitz syndrome (SLOS), congenital familial hypercholesterolemia (FH), and sitosterolemia (SITO). As expected, NP-C patients showed significant increase of both C-triol and 7-KC. Strong increase of both oxysterols was observed in NP-B and less pronounced in LAL deficiency. In SLOS, only 7-KC was markedly increased, whereas in both FH and in SITO, oxysterol concentrations were normal. Interestingly, in NP-C alone, we observed that plasma oxysterols correlate negatively with patient's age and positively with serum total bilirubin, suggesting the potential relationship between oxysterol levels and hepatic disease status. Our results indicate that oxysterols are reliable and sensitive biomarkers of NP-C.