Biomarkers to predict disease progression and therapeutic response in isolated methylmalonic acidemia.

Methylmalonic Acidemia (MMA) is a heterogenous group of inborn errors of metabolism caused by a defect in the methylmalonyl-CoA mutase (MMUT) enzyme or the synthesis and transport of its cofactor, 5'-deoxy-adenosylcobalamin. It is characterized by life-threatening episodes of ketoacidosis, chronic kidney disease, and other multiorgan complications. Liver transplantation can improve patient stability and survival and thus provides clinical and biochemical benchmarks for the development of hepatocyte-targeted genomic therapies. Data are presented from a US natural history protocol that evaluated subjects with different types of MMA including mut-type (N = 91), cblB-type (15), and cblA-type MMA (17), as well as from an Italian cohort of mut-type (N = 19) and cblB-type MMA (N = 2) subjects, including data before and after organ transplantation in both cohorts. Canonical metabolic markers, such as serum methylmalonic acid and propionylcarnitine, are variable and affected by dietary intake and renal function. We have therefore explored the use of the 1-13 C-propionate oxidation breath test (POBT) to measure metabolic capacity and the changes in circulating proteins to assess mitochondrial dysfunction (fibroblast growth factor 21 [FGF21] and growth differentiation factor 15 [GDF15]) and kidney injury (lipocalin-2 [LCN2]). Biomarker concentrations are higher in patients with the severe mut0 -type and cblB-type MMA, correlate with a decreased POBT, and show a significant response postliver transplant. Additional circulating and imaging markers to assess disease burden are necessary to monitor disease progression. A combination of biomarkers reflecting disease severity and multisystem involvement will be needed to help stratify patients for clinical trials and assess the efficacy of new therapies for MMA.

Comprehensive-targeted lipidomic analysis in Niemann-Pick C disease.

Niemann-Pick C disease (NPC) is a lysosomal disease caused by mutations in NPC1 or NPC2 genes responsible for intracellular accumulation of free cholesterol and glycosphingolipids in a variety of tissues. We collected plasma samples from 15 NPC1 patients and 15 age-matched controls to analyze the impairment of lipid metabolism. Comprehensive-targeted quantitative lipidomic analysis was per-formed by Ion Mobility Mass Spectrometry, while oxysterols and lyso-sphingolipids, the classical NPC biomarkers, were analyzed by LC-MS/MS. Lipidomic analysis allowed the quantitation of ~1100 lipid species, belonging to 13 different classes. Statistical analysis of collected data showed a significant differentiation between NPC patients and controls. Lipid profiling showed an elevation of arachidonic acid and total diacylglycerols. Conversely, sphingomyelins, phosphatidylethano-lamines, phosphatidylcholines, cholesterylesters, and lactosylceramides were decreased. Indeed, the lipid imbalance was consistent with the increased concentrations of oxysterols and lyso-sphingolipids. Our study revealed a novel disease biosignature suggesting new potential diagnostic biomarkers. The alteration in key lipids molecules involved in inflammatory pathways and in oxidative stress regulation, provides new insights in the complex pathophysiology of the disease, still largely un-known.

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.

The contribution of plasma oxysterols in the challenging diagnostic work-up of infantile cholestasis.

BACKGROUND: Infantile cholestasis (IC) is defined as an impairment of bile production or flow occurring in the first months of life. The diagnostic approach in IC is challenging since the differential diagnosis is broad. METHODS: We retrospectively evaluated 91 cholestatic infants referred to our department from 2014 to 2019. Patients with cholestasis underwent a complete IC diagnostic work-up including quantification of plasma oxysterols 7-ketocholesterol (7-KC) and cholestan-3ß,5α,6ß-triol (C-Triol). RESULTS: Oxysterols concentrations were mildly elevated in IC compared to control population. 7-KC and C-Triol plasma levels presented a linear relationship between them and with Spleen-Z score. Patients with NP-C showed the highest concentrations of both oxysterols compared with other etiologies of IC. Excluding NP-C patients, oxysterols concentrations were similar among all other etiological groups with no correlations found between them and the levels of cholesterol and bilirubin. ROC analysis identified AUCs of 1.0 for both oxysterols in predicting NP-C. CONCLUSION: Infants with IC should undergo a stepwise evaluation in which detailed clinical and deep analytical assessments are the main crossroads. Plasma oxysterols, a simple, reliable, and convenient diagnostic test should be included in the first steps of the diagnostic process in IC.

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.

Identification of bla(IMP-22) in Pseudomonas spp. in urban wastewater and nosocomial environments: biochemical characterization of a new IMP metallo-enzyme variant and its genetic location.

OBJECTIVES: The aim of the study was the biochemical characterization of a new variant of the metallo-beta-lactamase, IMP-22. Moreover, the genetic environment of the bla(IMP-22) gene was investigated in Pseudomonas fluorescens and Pseudomonas aeruginosa collected from urban wastewater and a teaching hospital in L'Aquila, Italy. METHODS: Molecular characterization of genetic elements was carried out by PCR and DNA sequencing methods. The new enzyme was purified from recombinant Escherichia coli BL21(DE)Rosetta/pBC-SK/IMP-22. Steady-state kinetic parameters (K(m) and V(max)) were determined for a large pattern of substrates. RESULTS: A new IMP metallo-beta-lactamase gene was found in a class 1 integron and in one case, in a plasmid of Pseudomonas spp. The bla(IMP-22) encodes for a pre-protein of 246 amino acids and the N-terminus of the mature beta-lactamase (NH(2)-PDLK) was also determined. The molecular mass and pI were 24 930 Da and 6.2, respectively. On the basis of the kinetic parameters calculated (K(m) and V(max)), IMP-22 was found to hydrolyse narrow- and extended-spectrum beta-lactams. Enzyme activity was found to be inhibited by metal chelators such as EDTA, 1,10-o-phenathroline and dipicolinic acid with an IC(50) of 800, 750 and 300 microM, respectively. CONCLUSIONS: The finding of the bla(IMP-22) gene in P. fluorescens environmental strains and P. aeruginosa clinical isolate suggests the ongoing spread of bla(MBL) genes in several bacterial species and in different environments.