Roberta Taurisano

A new simple and rapid LC–ESI-MS/MS method for quantification of plasma oxysterols as dimethylaminobutyrate esters. Its successful use for the diagnosis of Niemann–Pick type C disease

Two oxysterols, cholestan-3β,5α,6β-triol (C-triol) and 7-ketocholesterol (7-KC), have been recently proposed as diagnostic markers of Niemann-Pick type C (NP-C) disease, representing a potential alternative diagnostic tool to the more invasive and time consuming filipin test in cultured fibroblasts. Usually, the oxysterols are detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using atmospheric pressure chemical ionization (APCI) or electro-spray-ionization (ESI) sources, after a variety of derivatization procedures to enhance sensitivity. We developed a sensitive LC-MS/MS method to quantify the oxysterols in plasma as dimethylaminobutyrate ester, suitable for ESI analysis. This method, with an easy liquid-phase extraction and a short derivatization procedure, has been validated to demonstrate specificity, linearity, recovery, lowest limit of quantification, accuracy and precision. The assay was linear over a concentration range of 0.5-200ng/mL for C-triol and 1.0-200ng/mL for 7-KC. Intra-day and inter-day coefficients of variation (CV%) were <15% for both metabolites. Receiver operating characteristic analysis estimates that the area under curve was 0.998 for C-triol, and 0.972 for 7-KC, implying a significant discriminatory power for the method in this patient population of both oxysterols. In summary, our method provides a simple, rapid and non-invasive diagnostic tool for the biochemical diagnosis of NP-C disease.

Evaluation of plasma cholestane-3β,5α,6β-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-3β,5α,6β-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.

Wolman disease associated with hemophagocytic lymphohistiocytosis: attempts for an explanation.

The lysosomal acid lipase (LAL) is the enzyme responsible of the hydrolysis of cholesteryl esters and triglycerides within endo-lysosomes. Loss of enzyme activity leads to accumulation of cholesteryl esters and triglycerides in the lysosome of most tissues. The complete deficiency of LAL is responsible of Wolman disease (WD), a severe systemic disease manifesting in the first days of life with vomiting, diarrhea, failure to thrive, hepatosplenomegaly, jaundice, anemia, and thrombocytopenia. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition which may be genetically determined or secondary to infections, malignancies, immune deficiencies, and rheumatologic disorders. So far, some inborn errors of metabolism have been associated with HLH (e.g., lysinuric protein intolerance, Gaucher’s disease), and it has been anecdotally described in three WD patients, without any specific pathogenetic hypothesis. Here, we report on a WD patient, showing clear clinical, biochemical, and histological features indicative of HLH. We discuss the pathophysiological role of cholesteryl ester-induced inflammasome activation in macrophages, leading to a secondary HLH. Conclusion: This case indicates that WD can cause secondary HLH and suggests that a careful metabolic workup should be performed when facing to a pediatric patient with HLH.

When silence is noise: infantile-onset Barth syndrome caused by a synonymous substitution affecting TAZ gene transcription.

Barth syndrome (BTHS) is an X‐linked inborn error of metabolism which affects males. The main manifestations are cardiomyopathy, myopathy, hypotonia, growth delay, intermittent neutropenia and 3‐methylglutaconic aciduria. Diagnosis is confirmed by mutational analysis of the TAZ gene and biochemical dosage of the monolysocardiolipin/tetralinoleoyl cardiolipin (MLCL:L4‐CL) ratio. We report a 6‐year‐old boy who presented with severe hypoglycemia, lactic acidosis and severe dilated cardiomyopathy soon after birth. The MLCL:L4‐CL ratio confirmed BTHS (3.90 on patients fibroblast, normal: 0–0.3). Subsequent sequencing of the TAZ gene revealed only the new synonymous variant NM_000116.3 (TAZ):c.348C>T p.(Gly116Gly), which did not appear to affect the protein sequence. In silico prediction analysis suggested the new c.348C>T nucleotide change could alter the TAZ mRNA splicing processing. We analyzed TAZ mRNAs in the patients fibroblasts and found an abnormal skipping of 24 bases (NM_000116.3:c.346_371), with the consequent ablation of 8 amino acid residues in the tafazzin protein (NP_000107.1:p.Lys117_Gly124del). Molecular analysis of at risk female family members identified the patients sister and mother as heterozygous carriers. Apparently harmless synonymous variants in the TAZ gene can damage gene expression. Such findings widen our knowledge of molecular heterogeneity in BTHS.

Myasthenia gravis in a patient affected by glycogen storage disease type Ib: A further manifestation of an increased risk for autoimmune disorders?

SummaryGlycogen storage disease type Ib (GSD Ib, OMIM 232220) is an inborn disorder of glucose metabolism, caused by mutations in the G6PT gene, encoding a glucose 6-phosphate transporter (G6PT). GSD Ib is mainly associated with fasting hypoglycaemia and hepatomegaly. Most GSD Ib patients also show neutropenia and neutrophil dysfunction and therefore are at risk of developing severe infections and inflammatory bowel disease (IBD). An increased risk for autoimmune disorders, such as thyroid autoimmunity and Crohn-like disease, has also been demonstrated, but no systematic study on the prevalence of autoimmune disorders in GSD Ib patients has ever been performed. We describe a 25-year-old patient affected by GSD Ib who developed ‘seronegative’ myasthenia gravis (MG), presenting with bilateral eyelid ptosis, diplopia, dysarthria, severe dysphagia, dyspnoea and fatigue. The repetitive stimulation of peripheral nerves test showed signs of exhaustion of neuromuscular transmission, particularly evident in the cranial area. Even in the absence of identifiable anti-acetylcholine receptor antibodies, seronegative MG is considered an autoimmune disorder and may be related to the disturbed immune function observed in GSD Ib patients.

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

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.

3-Methylglutaconic aciduria, a frequent but underrecognized finding in carbamoyl phosphate synthetase I deficiency

The urea cycle disorder carbamoyl phosphate synthetase I deficiency is an important differential diagnosis in the encephalopathic neonate. This intoxication type inborn error of metabolism often leads to neonatal death or severe and irreversible damage of the central nervous system, even despite appropriate treatment. Timely diagnosis is crucial, but can be difficult on routine metabolite level. Here, we report ten neonates from eight families (finally) diagnosed with CPS1 deficiency at three tertiary metabolic centres. In seven of them the laboratory findings were dominated by significantly elevated urinary 3-methylglutaconic acid levels which complicated the diagnostic process. Our findings are both important for the differential diagnosis of patients with urea cycle disorders and also broaden the differential diagnosis of hyperammonemia associated with 3-methylglutaconic aciduria, which was earlier only reported in TMEM70 and SERAC1 defect.

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.

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

BackgroundMucopolysaccharidosis-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.MethodsIn 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.ResultsDifferent 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.ConclusionsWe 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.

Does The Frequency of Enzymatic Replacement Therapy Influence Patients’ Level of Adaptive Functioning?

Background Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic disorders that result from defects in lysosomal function. LSDs are generally multisystemic diseases with reduced life expectancy. Enzymatic replacement therapy (ERT) is available for some LSDs and has led to an improvement of many somatic symptoms of some metabolic disorders. To our best knowledge no study has explored the impact of ERT frequency on adaptive functioning in this unique pediatric population. We hereby present a pilot study. Methods The parents of 27 patients with LSDs, aged between six months and 16 years (mean age 9 years 4 months), were asked to complete the Vineland Adaptive Behavior Scales (VABS) Survey Form interview to measure children’s functional outcomes within four domains: communication, daily living, socialization and motor ability. Disorders present amongst the sample included MPS (N=13; I, I H-S, I H, I S, II, IV-Morquio, VI) Gaucher (N=6; types I and III) and Pompe (N=8; infantile and late onset forms). Data collection took place during patients’ visits to the Bambino Gesu Children’s Hospital in Rome (Italy) for ERT treatment. Results The total sample obtained significantly lower scores ( When considering only children with an IQ in the normal ranges (>85; N=21), no significant differences were found comparing children’s adaptive functioning by splitting the sample into high and low ERT frequency (high=once a week; low=fortnightly). However, 58% of patients undergoing ERT once a week and 43% of patients undergoing ERT fortnightly presented with scores below the normal ranges ( Discussion Despite children with good cognitive capacities who did not differ significantly on level of adaptive functioning based on high or low ERT frequency, results suggest that higher frequency of treatment was associated with a higher occurrence of scores below the normal ranges in the adaptive behavior in this sample. Investigating this tendency could be of crucial importance in understanding whether lower scores in the daily living skills domain of the VABS and, more in general, maladaptive functioning might be associated with ERT frequency. Finally, a clearer understanding of the role of treatment frequency in this population’s adaptive functioning can lead to an improvement of both patients and families’ health outcomes.