Proteomics in Inherited Metabolic Disorders.

Inherited metabolic disorders (IMD) are rare medical conditions caused by genetic defects that interfere with the body's metabolism. The clinical phenotype is highly variable and can present at any age, although it more often manifests in childhood. The number of treatable IMDs has increased in recent years, making early diagnosis and a better understanding of the natural history of the disease more important than ever. In this review, we discuss the main challenges faced in applying proteomics to the study of IMDs, and the key advances achieved in this field using tandem mass spectrometry (MS/MS). This technology enables the analysis of large numbers of proteins in different body fluids (serum, plasma, urine, saliva, tears) with a single analysis of each sample, and can even be applied to dried samples. MS/MS has thus emerged as the tool of choice for proteome characterization and has provided new insights into many diseases and biological systems. In the last 10 years, sequential window acquisition of all theoretical fragmentation spectra mass spectrometry (SWATH-MS) has emerged as an accurate, high-resolution technique for the identification and quantification of proteins differentially expressed between healthy controls and IMD patients. Proteomics is a particularly promising approach to help obtain more information on rare genetic diseases, including identification of biomarkers to aid early diagnosis and better understanding of the underlying pathophysiology to guide the development of new therapies. Here, we summarize new and emerging proteomic technologies and discuss current uses and limitations of this approach to identify and quantify proteins. Moreover, we describe the use of proteomics to identify the mechanisms regulating complex IMD phenotypes; an area of research essential to better understand these rare disorders and many other human diseases.

Utility of Gene Panels for the Diagnosis of Inborn Errors of Metabolism in a Metabolic Reference Center.

Next-generation sequencing (NGS) technologies have been proposed as a first-line test for the diagnosis of inborn errors of metabolism (IEM), a group of genetically heterogeneous disorders with overlapping or nonspecific phenotypes. Over a 3-year period, we prospectively analyzed 311 pediatric patients with a suspected IEM using four targeted gene panels. The rate of positive diagnosis was 61.86% for intermediary metabolism defects, 32.84% for complex molecular defects, 19% for hypoglycemic/hyperglycemic events, and 17% for mitochondrial diseases, and a conclusive molecular diagnosis was established in 2-4 weeks. Forty-one patients for whom negative results were obtained with the mitochondrial diseases panel underwent subsequent analyses using the NeuroSeq panel, which groups all genes from the individual panels together with genes associated with neurological disorders (1870 genes in total). This achieved a diagnostic rate of 32%. We next evaluated the utility of a tool, Phenomizer, for differential diagnosis, and established a correlation between phenotype and molecular findings in 39.3% of patients. Finally, we evaluated the mutational architecture of the genes analyzed by determining z-scores, loss-of-function observed/expected upper bound fraction (LOEUF), and haploinsufficiency (HI) scores. In summary, targeted gene panels for specific groups of IEMs enabled rapid and effective diagnosis, which is critical for the therapeutic management of IEM patients.

Diagnosis of Mucopolysaccharidoses and Mucolipidosis by Assaying Multiplex Enzymes and Glycosaminoglycans.

Mucopolysaccharidoses (MPS) and mucolipidosis (ML II/III) are a group of lysosomal storage disorders (LSDs) that occur due to a dysfunction of the lysosomal hydrolases responsible for the catabolism of glycosaminoglycans (GAGs). However, ML is caused by a deficiency of the enzyme uridine-diphosphate N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. A timely diagnosis of MPS and ML can lead to appropriate therapeutic options for patients. To improve the accuracy of diagnosis for MPS and ML in a high-risk population, we propose a combination method based on known biomarkers, enzyme activities, and specific GAGs. We measured five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of heparan sulfate (HS), dermatan sulfate (DS), and two kinds of keratan sulfate (KS)) in dried blood samples (DBS) to diagnose suspected MPS patients by five-plex enzyme and simultaneous five GAGs assays. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both assays. These combined assays were tested for 43 patients with suspected MPS and 103 normal control subjects. We diagnosed two MPS I, thirteen MPS II, one MPS IIIB, three MPS IVA, two MPS VI, and six ML patients with this combined method, where enzymes, GAGs, and clinical manifestations were compatible. The remaining 16 patients were not diagnosed with MPS or ML. The five-plex enzyme assay successfully identified MPS patients from controls. Patients with MPS I, MPS II, and MPS IIIB had significantly elevated HS and DS levels in DBS. Compared to age-matched controls, patients with ML and MPS had significantly elevated mono-sulfated KS and di-sulfated KS levels. The results indicated that the combination method could distinguish these affected patients with MPS or ML from healthy controls. Overall, this study has shown that this combined method is effective and can be implemented in larger populations, including newborn screening.

Plasma Proteomic Analysis in Morquio A Disease.

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal disease caused by mutations in the gene encoding the enzymeN-acetylgalactosamine-6-sulfate sulfatase (GALNS), and is characterized by systemic skeletal dysplasia due to excessive storage of keratan sulfate (KS) and chondroitin-6-sulfate in chondrocytes. Although improvements in the activity of daily living and endurance tests have been achieved with enzyme replacement therapy (ERT) with recombinant human GALNS, recovery of bone lesions and bone growth in MPS IVA has not been demonstrated to date. Moreover, no correlation has been described between therapeutic efficacy and urine levels of KS, which accumulates in MPS IVA patients. The objective of this study was to assess the validity of potential biomarkers proposed by other authors and to identify new biomarkers. To identify candidate biomarkers of this disease, we analyzed plasma samples from healthy controls (n=6) and from untreated (n=8) and ERT-treated (n=5, sampled before and after treatment) MPS IVA patients using both qualitative and quantitative proteomics analyses. The qualitative proteomics approach analyzed the proteomic profile of the different study groups. In the quantitative analysis, we identified/quantified 215 proteins after comparing healthy control untreated, ERT-treated MPSIVA patients. We selected a group of proteins that were dysregulated in MPS IVA patients. We identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: fetuin-A, vitronectin, alpha-1antitrypsin, and clusterin. Further studies of cartilage and bone samples from MPS IVA patients will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.

Newborn screening for Fabry disease in the north-west of Spain.

Fabry disease is an X-linked lysosomal storage disorder caused by the impairment of α-galactosidase A. Enzyme replacement therapy is available to treat patients, who often experience delayed diagnosis. A newborn screening for Fabry disease was performed to study the prevalence of the pathology and to evaluate the possibility to implement the test in systematic screenings. We collected 14,600 dried blood spot samples (7575 males and 7025 females) and carried out a diagnostic study by fluorometric measurement of α-galactosidase A enzymatic activity and GLA gene sequencing. We detected one patient with a mutation in GLA associated with classical Fabry Disease (M290I), ten subjects carrying genetic variants of uncertain diagnosis (S126G, R118C, A143T), and a girl with the non-characterized variant F18Y, which was not previously described. Additional 25 samples presented nucleotide substitutions described as polymorphisms (D313Y, rs2071225, and rs2071397). The estimated prevalence for Fabry disease in north-western Spanish males is of 0.013%. CONCLUSION: These results confirm that the prevalence of Fabry disease is underestimated and systematic screening is feasible; however, further characterization of variants of uncertain clinical significance is necessary to establish protocols of patients' management. What is Known: • Fabry disease is a rare disease of delayed diagnosis, whose prevalence is underestimated. However, early diagnosis is important for better efficiency of the current available treatment. What is New: • This newborn screening for Fabry disease performed on Spanish population reveals a prevalence of genetic alterations in GLA of 0.1% in males (0.013% with classic Fabry disease) and also characterizes these modifications in order to discriminate between pathogenic mutations and genetic variants of unknown significance.

TiS3 transistors with tailored morphology and electrical properties.

Control over the morphology of TiS3 is demonstrated by synthesizing 1D nanoribbons and 2D nanosheets. The nanosheets can be exfoliated down to a single layer. Through extensive characterization of the two morphologies, differences in the electronic properties are found and attributed to a higher density of sulphur vacancies in nanosheets, which, according to density functional theory calculations, leads to an n-type doping.