The diagnostic challenge of mild citrulline elevation at newborn screening.

Citrulline is a target analyte measured at expanded newborn screening (NBS) and its elevation represents a biomarker for distal urea cycle disorders and citrin deficiency. Altered ratios of citrulline with other urea cycle-related amino acids are helpful for the differential diagnosis. However, the use of cut-off values in screening programmes has raised the issue about the interpretation of mild elevation of citrulline levels detected at NBS, below the usual range observed in the "classical/severe" forms of distal urea cycle disorders and in citrin deficiency. Herein, we report ten subjects with positive NBS for a mild elevation of citrulline (<100 µmol/L), in whom molecular investigations revealed carriers status for argininosuccinate synthase deficiency, a milder form of argininosuccinate lyase deficiency and two other diseases, lysinuric protein intolerance and dihydrolipoamide dehydrogenase deficiency, not primarily affecting the urea cycle. To guide the diagnostic process, we have designed an algorithm for mild citrulline elevation (<100 µmol/L) at NBS, which expands the list of disorders to be included in the differential diagnosis.

Ethylmalonic encephalopathy and liver transplantation: long-term outcome of the first treated patient.

BACKGROUND: Ethylmalonic encephalopathy (EE) is a severe intoxication-type metabolic disorder with multisystem clinical features and leading to early death. In 2014, based on the promising results obtained by liver-targeted gene therapy in Ethe1-/- mouse model, we successfully attempted liver transplantation in a 9-month-old EE girl. Here we report her long-term follow-up, lasting over 6 years, with a comprehensive evaluation of clinical, instrumental and biochemical assessments. RESULTS: Neurological signs initially reverted, with a clinical stabilization during the entire follow-up course. Accordingly, gross motor functions improved and then stabilized. Psychomotor evaluations documented an increasing communicative intent, the acquisition of new social skills and the capability to carry out simple orders. Neurophysiological assessments, which included EEG, VEP/ERG and BAEPs, remained unchanged. Brain MRI also stabilized, showing no further lesions and cerebral atrophy improvement. Compared to pre-transplant assessments, urinary ethylmalonic acid strikingly reduced, and plasma thiosulphate fully normalized. The child maintained good clinical conditions and never experienced metabolic crises nor epileptic seizures. CONCLUSIONS: The long-term follow-up of the first EE transplanted patient demonstrates that liver transplantation stabilizes, or even improves, disease course, therefore representing a potentially elective option especially in early-diagnosed patients, such as those detected by newborn screening, before irreversible neurological damage occurs.

A new UHPLC-MS/MS method for the screening of urinary oligosaccharides expands the detection of storage disorders.

BACKGROUND: Oligosaccharidoses are storage disorders due to enzymatic defects involved in the breakdown of the oligosaccharidic component of glycosylated proteins. The defect cause the accumulation of oligosaccharides (OS) and, depending on the lacking enzyme, results in characteristic profiles which are helpful for the diagnosis. We developed a new tandem mass spectrometry method for the screening of urinary OS which was applied to identify a large panel of storage disorders. METHODS: The method was set-up in urine and dried urine spots (DUS). Samples were analysed, without derivatization and using maltoheptaose as internal standard, by UHPLC-MS/MS with MRM acquisition of target OS transitions, including Glc4, the biomarker of Pompe disease. The chromatographic run was < 30 min. Samples from patients with known storage disorders were used for clinical validation. RESULTS: The method allowed to confirm the diagnosis of oligosaccharidoses (sialidosis, α-/ß-mannosidosis, fucosidosis, aspartylglucosaminuria) and of GM1 and GM2 (Sandhoff type) gangliosidosis, by detecting specific OS profiles. In other storage disorders (mucolipidosis II and III, mucopolysaccharidosis type IVB) the analyisis revealed abnormal OS excretion with non-specific profiles. Besides Pompe disease, the tetrasaccharide Glc4 was increased also in disorders of autophagy (Vici syndrome, Yunis-Varon syndrome, and Danon disease) presenting cardiomuscular involvement with glycogen storage. Overall, results showed a clear separation between patients and controls, both in urine and in DUS. CONCLUSION: This new UHPLC/MS-MS method, which is suitable for rapid and easy screening of OS in urine and DUS, expands the detection of storage disorders from oligosaccharidoses to other diseases, including the novel category of inherited disorders of autophagy.

Delineating the neurological phenotype in children with defects in the ECHS1 or HIBCH gene.

The neurological phenotype of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) and short-chain enoyl-CoA hydratase (SCEH) defects is expanding and natural history studies are necessary to improve clinical management. From 42 patients with Leigh syndrome studied by massive parallel sequencing, we identified five patients with SCEH and HIBCH deficiency. Fourteen additional patients were recruited through collaborations with other centres. In total, we analysed the neurological features and mutation spectrum in 19 new SCEH/HIBCH patients. For natural history studies and phenotype to genotype associations we also included 70 previously reported patients. The 19 newly identified cases presented with Leigh syndrome (SCEH, n = 11; HIBCH, n = 6) and paroxysmal dystonia (SCEH, n = 2). Basal ganglia lesions (18 patients) were associated with small cysts in the putamen/pallidum in half of the cases, a characteristic hallmark for diagnosis. Eighteen pathogenic variants were identified, 11 were novel. Among all 89 cases, we observed a longer survival in HIBCH compared to SCEH patients, and in HIBCH patients carrying homozygous mutations on the protein surface compared to those with variants inside/near the catalytic region. The SCEH p.(Ala173Val) change was associated with a milder form of paroxysmal dystonia triggered by increased energy demands. In a child harbouring SCEH p.(Ala173Val) and the novel p.(Leu123Phe) change, an 83.6% reduction of the protein was observed in fibroblasts. The SCEH and HIBCH defects in the catabolic valine pathway were a frequent cause of Leigh syndrome in our cohort. We identified phenotype and genotype associations that may help predict outcome and improve clinical management.

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.

Clinical and Biochemical Features in a Patient With Mitochondrial Fission Factor Gene Alteration.

Mitochondrial Fission Factor (MFF) is part of a protein complex that promotes mitochondria and peroxisome fission. Hitherto, only 5 patients have been reported harboring mutations in MFF, all of them with the clinical features of a very early onset Leigh-like encephalopathy. We report on an 11-year-old boy with epileptic encephalopathy. He presented with neurological regression, epileptic myoclonic seizures, severe intellectual disability, microcephaly, tetraparesis, optic atrophy, and ophthalmoplegia. Brain MRI pattern was compatible with Leigh syndrome. NGS-based analysis of a gene panel for mitochondrial disorders revealed a homozygous c.892C>T (p. Arg298*) in the MFF gene. Fluorescence staining detected abnormal morphology of mitochondria and peroxisomes in fibroblasts from the patient; a strong reduction in MFF protein levels and the presence of truncated forms were observed. No biochemical alterations denoting peroxisomal disorders were found. As reported in other disorders affecting the dynamics of intracellular organelles, our patient showed clinical features suggesting both mitochondrial and peroxisomal impairment. High levels of lactate in our case suggested an involvement of the energetic metabolism but without clear respiratory chain deficiency, while biomarkers of peroxisomal dysfunction were normal. We confirm that MFF mutations are associated with epileptic encephalopathy with Leigh-like MRI pattern.

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.

The impact of biomarkers analysis in the diagnosis of Niemann-Pick C disease and acid sphingomyelinase deficiency.

BACKGROUND: Although representing two distinct disease entities, Niemann-Pick disease type C (NP-C) disease and acid sphingomyelinase deficiency (ASMD) share several phenotypic features. The lack of biomarkers was responsible in the past of diagnostic delay. Recently, plasma oxysterols, cholestan-3ß,5α,6ß-triol (Triol) and 7-ketocholesterol (7-KC) and lysosphingolipids, Lyso-sphingomyelin (Lyso-SM) and Lysosphingomyelin-509 (Lyso-SM-509), have been proposed as diagnostic biomarkers. We aimed to assess the diagnostic power of the two biomarkers categories and to evaluate possible correlations with patients' age and clinical phenotypes. PATIENTS AND METHODS: We analyzed plasma oxysterols and lysosphingolipids in patients affected by NP-C and ASMD, and compared with healthy controls. RESULTS: Oxysterols were always increased in both NP-C and ASMD. In NP-C, Lyso-SM and Lyso-SM-509 were increased in 70%, and 100% of patients, respectively. Biomarkers negatively correlated with patients' age, with highest levels in early-infantile, intermediate in the late-infantile and lowest in the juvenile phenotype. In ASMD, lysosphingolipids were both increased, with a greater order of magnitude than in NP-C, with highest levels in chronic-neurovisceral vs visceral phenotype. CONCLUSIONS: Lysosphingolipids are useful biomarkers for a rapid and precise diagnosis, allowing clear distinction between NP-C and ASMD. They are more reliable biomarkers than oxysterols and correlate with patients' age and clinical phenotype.

The urinary organic acids profile in single large-scale mitochondrial DNA deletion disorders.

Single large-scale mitochondrial DNA deletions disorders are classified into three main phenotypes with frequent clinical overlap: Pearson marrow-pancreas syndrome (PMS), Kearns-Sayre syndrome (KSS) and chronic progressive external ophtalmoplegia (PEO). So far, only few anecdotal studies have reported on the urinary organic acids profile in this disease class. In this single-center retrospective study, we performed quantitative evaluation of urinary organic acids in a series of 15 pediatric patients, 7 with PMS and 8 with KSS. PMS patients showed an organic acids profile almost constantly altered, whereas KSS patients frequently presented with normal profiles. Lactate, 3-hydroxybutyrate, 3-hydroxyisobutyrate, fumarate, pyruvate, 2-hydroxybutyrate, 2-ethyl-3-hydroxypropionate, and 3-methylglutaconate represented the most frequent metabolites observed in PMS urine. We also found novel metabolites, 3-methylglutarate, tiglylglycine and 2-methyl-2,3-dihydroxybutyrate, so far never reported in this disease. Interestingly, patients with a disease onset as PMS evolving overtime into KSS phenotype, presented persistent and more pronounced alterations of organic acid signature than in patients with a pure KSS phenotype. Our study shows that the quantitative analysis of urinary organic acid profile represents a helpful tool for the diagnosis of PMS and for the differential diagnosis with other inherited diseases causing abnormal organic acidurias.

Association between Maternal and Foetal Erythrocyte Fatty Acid Profiles and Birth Weight.

Regular foetal development is crucial for assuring good health status in the offspring. The quality and quantity of maternal dietary fatty acids (FAs) can affect growth. The study aimed to: (1) investigate the association of maternal/foetal lipid profiles with birth weight (BW); and (2) compare these profiles in small, appropriate, and large for gestational age (SGA, AGA, and LGA) infants. FAs were measured in erythrocyte membranes using gas chromatography analysis in 607 mother-infant pairs (316 males, 52.1%). In the quantile regression, a significant association between BW and levels of maternal linoleic acid (LA; C18:2, n-6; coefficient: 18.66; p = 0.010), arachidonic acid (AA; C20:4, n-6; coefficient: 11.35; p = 0.007), docosahexaenoic acid (DHA; C22:6, n-3; coefficient: 29.73; p = 0.007), polyunsaturated FAs (coefficient: 8.55; p = 0.001), foetal DHA (coefficient: -22.82; p = 0.037), and saturated FAs (coefficient: -65.41; p = 0.002) was found. Myristic (C14:0) and pentadecanoic acids (C15:0), both maternal (p = 0.000; p = 0.017) and foetal (p = 0.009; p = 0.002), and maternal erucic acid (C22:1, n-9; p = 0.026) were found at higher levels in SGA infants as compared to AGA ones. Conversely, maternal LA, AA, and omega 6 FAs levels were higher in AGA infants (p = 0.037; p = 0.003; p = 0.026, respectively). Maternal and foetal polyunsaturated and omega 6 FAs levels are positively related to BW, while a lipid profile rich in saturated FAs and erucic acid may influence the risk of SGA.

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.

Expanding the molecular diversity and phenotypic spectrum of glycerol 3-phosphate dehydrogenase 1 deficiency.

Transient infantile hypertriglyceridemia (HTGT1; OMIM #614480) is a rare autosomal recessive disorder, which manifests in early infancy with transient hypertriglyceridemia, hepatomegaly, elevated liver enzymes, persistent fatty liver and hepatic fibrosis. This rare clinical entity is caused by inactivating mutations in the GPD1 gene, which encodes the cytosolic isoform of glycerol-3-phosphate dehydrogenase. Here we report on four patients from three unrelated families of diverse ethnic origins, who presented with hepatomegaly, liver steatosis, hypertriglyceridemia, with or without fasting ketotic hypoglycemia. Whole exome sequencing revealed the affected individuals to harbor deleterious biallelic mutations in the GPD1 gene, including the previously undescribed c.806G > A (p.Arg269Gln) and c.640T > C (p.Cys214Arg) mutations. The clinical features in three of our patients showed several differences compared to the original reports. One subject presented with recurrent episodes of fasting hypoglycemia along with hepatomegaly, hypetriglyceridemia, and elevated liver enzymes; the second showed a severe liver disease, with intrahepatic cholestasis associated with kidney involvement; finally, the third presented persistent hypertriglyceridemia at the age of 30 years. These findings expand the current knowledge of this rare disorder, both with regard to the phenotype and molecular basis. The enlarged phenotypic spectrum of glycerol-3-phosphate dehydrogenase 1 deficiency can mimic other inborn errors of metabolism with liver involvement and should alert clinicians to recognize this entity by considering GPD1 mutations in appropriate clinical settings.

A new multiplex method for the diagnosis of peroxisomal disorders allowing simultaneous determination of plasma very-long-chain fatty acids, phytanic, pristanic, docosahexaenoic and bile acids by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Peroxisomal disorders (PDs) present with wide phenotypic variability. An appropriate diagnosis requires a complete analysis of peroxisomal metabolites. We developed a multiplex LC-MS/MS method, using atmospheric pressure chemical ionization allowing the simultaneous determination in plasma of very-long-chain fatty acids, phytanic, pristanic, docosahexaenoic acids and di- and tri-hydroxycolestanoic bile acids. Two hundred microliters of plasma extracted with acetonitrile and 200µl extracted with hexane after an acid hydrolysis were combined, evaporated, dissolved in 10µl of methanol and analyzed. The acquisition was in negative-ion mode using multiple reaction monitoring. The method was validated analytically and clinically. Linearity was 0.1-200µmol/l for docosanoic, cis-13-docosenoic, tetracosanoic, cis-15-tetracosenoic and phytanic acids; 0.01-10µmol/l for hexacosanoic acid; 0.02-20µmol/l for di-hydroxycolestanoic, tri-hydroxycolestanoic and pristanic acids; 0.3-300µmol/l for docosahexaenoic acid. Intra-day and inter-day CVs were below 3.88 and 3.98 respectively for all compounds. Samples from patients with known peroxisomal disorders were compared with controls and the method allowed to confirm the diagnosis in all subjects with a 100% sensitivity. The advantage of this multiplex method is to allow in a single chromatographic run the simultaneous determination of a large number of peroxisome biomarkers with a simple preparative phase without derivatization.

Determination of plasma pipecolic acid by an easy and rapid liquid chromatography-tandem mass spectrometry method.

Pipecolic acid (PA) is an important biochemical marker for the diagnosis of peroxisomal disorders. PA is also a factor responsible for hepatic encephalopathy and a possible biomarker for pyridoxine-dependent seizures. We developed an easy and rapid PA quantification method, by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), requiring no derivatization and applicable to small sample volumes. Plasma (100 µl) is extracted with 500 µl acetonitrile (ACN) containing 2 µmol/l [(2)H5]-phenylalanine as internal standard, vortexed and centrifuged. The supernatant is analyzed by HPLC-MS/MS in positive-ion mode using multiple reaction monitoring scan type. HPLC column is a Luna HILIC (150×3.0mm; 3 µ 200A): Buffer A: ammonium formate 5 mmol/l; Buffer B: ACN/H20 90:10 containing ammonium formate 5 mmol/l. PA retention time is 4.86 min. Recovery was 93.8%, linearity was assessed between 0.05 and 50 µmol/l (R(2)=0.998), lower limit of detection was 0.010 µmol/l and lower limit of quantification was 0.050 µmol/l. Coefficient of variation was 3.2% intra-assay and 3.4% inter-assay, respectively. Clinical validation was obtained by comparing PA plasma values from 5 patients affected by peroxisomal disorders (mean, 23.38 µmol/l; range, 11.20-37.1 µmol/l) to 24 ages related healthy subjects (mean, 1.711 µmol/l; range, 0.517-3.580 µmol/l).

Recently discovered adipokines and cardio-metabolic comorbidities in childhood obesity.

White adipose tissue (WAT) asset, in terms of cell number, fat storage capacity and endocrine function, is largely determined in early stages of life and is pivotal for shaping the WAT pro-inflammatory behavior. WAT derived adipokines have been shown to play a main role in several cardio-metabolic abnormalities of obesity. This review focuses on the most recently identified adipokines, namely adipocyte-fatty acid-binding protein, chemerin, fibroblast growth factor-21, lipocalin-2, omentin-1 and vaspin; their role in the pathogenesis of obesity and associated cardio-metabolic abnormalities; and on their adaptive response to body weight change. Evidence consistently suggests a pathogenic role for A-FABP, chemerin and FGF-21. Nevertheless, large population studies are needed to verify whether they can be useful to predict the risk of cardio-metabolic abnormalities in adulthood and/or monitor the clinical response to therapeutic interventions.