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WO2013030785A1 - Biomarqueurs de troubles du stockage lysosomal - Google Patents

Biomarqueurs de troubles du stockage lysosomal Download PDF

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Publication number
WO2013030785A1
WO2013030785A1 PCT/IB2012/054469 IB2012054469W WO2013030785A1 WO 2013030785 A1 WO2013030785 A1 WO 2013030785A1 IB 2012054469 W IB2012054469 W IB 2012054469W WO 2013030785 A1 WO2013030785 A1 WO 2013030785A1
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sample
metallothionein
protein
disease
mld
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PCT/IB2012/054469
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English (en)
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Alessandra Biffi
Martina CESANI
Clemens R. Scherzer
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Ospedale San Raffaele Srl
Fondazione Telethon
The Brigham And Women's Hospital, Inc.
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Publication of WO2013030785A1 publication Critical patent/WO2013030785A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/825—Metallothioneins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/825—Metallothioneins

Definitions

  • the present invention relates to methods useful in diagnosis and prognosis of a lysosomal storage disorder by determining the expression levels of one or more metallothioneins.
  • the present invention also relates to a method of monitoring the progress of a lysosomal storage disorder and its response to therapy by determining the expression levels of one or more metallothioneins.
  • Lysosomal Storage Disorders comprise a class of inherited diseases characterized by disruption of normal lysosomal function resulting in the accumulation of incompletely degraded substrates that have been targeted for degradation after endocytosis or autophagy.
  • the ensuing accumulation of the substrate itself or of the product(s) of an alternative metabolic route in lysosomes affects the architecture and function of the cells, leading to cell dysfunction or death. Further, the primary defect is frequently exacerbated by secondary responses. This is of particular relevance in the Central Nervous System (CNS) where neuroinflammation occurs representing a primary reaction to substrate accumulation within microglia and astrocytes and/or an inflammatory response to primary neuronal or oligodendroglial damage.
  • CNS Central Nervous System
  • Metachromatic Leukodystrophy a demyelinating LSD due to mutations in the Arylsulfatase A (ARSA) gene (Aicardi (1998) In Disease of the nervous system in childhood. Cambridge University Press; Kolodny and Neudorfer (2003) Metachromatic Leukodistrophy and Multiple Sulfatase Deficiency: Sulfatide Lipidosis. 3rd ed, Heinemann) is a prototypical example of LSD with progressive accumulation of un-degraded substrates in the nervous system and secondary neuroinflammation and degeneration.
  • Clinical manifestations consisting of severe and unrelenting motor and cognitive impairment, and disease progression are more severe in the early onset clinical variants, leading to death usually within the first decade of life.
  • a correlation between the phenotype of MLD patients and the type of ARSA mutation they bear has recently been demonstrated (Cesani et al. (2009) Hum Mutat, 30, E936-945; Biffi. et al. (2008) Clinical Genetics, 74, 349-357).
  • LSDs are rare diseases with variable phenotypes and unpredictable progression over long periods. Accordingly, there is an urgent need for identifying biomarkers associated with LSDs and which can be used to provide improved methods of diagnosing LSDs. Furthermore, considerable research activity is currently focused on developing strategies to target LSDs with CNS involvement. Gene therapy (Biffi et al. (2004) J. Clin. Invest., 113, 1118-1129; Biffi et al. (2006) J Clin. Invest., 116, 3070- 3082), enzyme replacement therapy (Matzner et al. (2005) Hum Mol Genet, 14, 1139- 1152; Matzner et al.
  • a method of diagnosis or prognosis of a lysosomal storage disorder in a subject wherein said method comprises
  • An increase in the expression level of metallothionein in a sample relative to a sample from a healthy subject may be indicative of a lysosomal storage disorder.
  • a lysosomal storage disorder in a subject, wherein said method comprises:
  • the finding of expression levels of metallothionein of at least about 1.5, 2, 5, 10 or 50 fold relative to a healthy subject may indicate the presence of an LSD.
  • the finding of expression levels of metallothionein of at least about 1.5, 2, 2.5, 3, 4 or 5 fold relative to a healthy subject when measuring metallothionein mRNA abundance in T-cells/mononuclear cells may indicate the presence of an LSD.
  • the finding of expression levels of metallothionein of at least about 10, 15 or 20 fold relative to a healthy subject when measuring metallothionein mRNA abundance in brain samples or nerve biopsies may indicate the presence of a lysosomal storage disorder.
  • a lysosomal storage disorder comprising the steps of:
  • An increase in metallothionein expression in the second sample relative to the first sample indicates disease progression.
  • a decrease in metallothionein expression in the second sample relative to the first sample indicates amelioration of the symptoms.
  • the second sample is obtained from the subject at least 1, 2, 5, 10, 20, 50, 100, 200 or 300 days after the first sample.
  • the second aspect of the invention may be used to momtor the progress of an LSD in response to treatment (e.g., by bone marrow transplantation, gene therapy treatment, enzyme replacement therapy) of the LSD.
  • the present invention may be used to assess the effectiveness of an LSD treatment.
  • the treatment may be administered between the taking of the first sample and the taking of the second sample from the patient.
  • the sample is nervous tissue, 3 ⁇ 4lood (including blood cells and plasma/serum) or cerebrospinal fluid.
  • the sample is a population of T cells.
  • the metallothionein expression levels may be determined by, for example, quantitative RT-PCR.
  • the metallothionein expression levels may be determined by an immunoassay such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunofluorescence assay (IF A), enzyme linked assay (EI A), luminescence immunoassay (LI A), or Western Blot (WB).
  • the immunoassay may use an antibody immunospecifc for the metallothionein to detect metallothionein protein levels.
  • the metallothionein protein levels may be determined also by HPLC and mass spectrometry (Monicou et al. (2010) Anal Chem 82 6947-57).
  • the metallothionein may be selected from the group consisting of MT1, MT2, MT3 and MT4.
  • the metallothionein is selected from the group consisting of MT1 and MT2.
  • the metallothionein is selected from the group consisting of MT1A, MT1E, MT1F, MT2, MT3 and MT4. In another embodiment, the metallothionein is selected from the group consisting of MT1A, MT1E and MT2A. In a further embodiment, the metallothionein is selected from the group consisting of MT1 A, MT1E, MT2A and MTE.
  • the methods of the present invention may comprise deternii ing the expression level of more than one metallothionein.
  • the method comprises determining the expression levels of MT1A, MT1E and MT2A.
  • the method comprises determining the expression levels of MT1A, MT1E, MT2A and/or MTE.
  • the methods of the present invention may identify the expression levels of metallothionein using antibodies raised against the general metallothionein protein.
  • an antibody directed against a metallothionein for use in a diagnostic for a lysosomal storage disorder.
  • the lysosomal storage disorder is selected from the group consisting of Metachromatic Leukodystrophy (MLD), Globoid Cell Leukodystrophy (GLD), Mucopolysaccharidosis type I (MPS I), Mucopolysaccharidosis type III (MPS III), Neimann Pick disease (NPC), Neuronal Ceroidolipofuscinosis (NCL) and Sandhoff disease (SD).
  • MLD Metachromatic Leukodystrophy
  • GLD Globoid Cell Leukodystrophy
  • MPS I Mucopolysaccharidosis type I
  • MPS III Mucopolysaccharidosis type III
  • NPC Neimann Pick disease
  • NCL Neuronal Ceroidolipofuscinosis
  • SD Sandhoff disease
  • the lysosomal storage disorder is MLD.
  • the subject is a human subject.
  • MTs Metallothionerns
  • MTs are a family of small (-6-7 kDa), heat-resistant proteins containing 25-30% cysteine residues that are evolutionarily highly conserved in a broad range of species from yeast to mammals.
  • MTs are up-regulated by glucocorticoids, oxidative stress and a variety of heavy metals, such as copper, cadmium, mercury and zinc (Andrews (2000) Biochem. Pharmacol. 59, 95-104).
  • Isoforms range from MT-1 to MT-4 and have slightly different amino acid composition.
  • MTs bind metals and protect against their toxicity, as was first demonstrated in aquatic species, such as fish, arthropods and molluscs from contaminated waters.
  • MTs are considered to act as antioxidants, although by undetermined mechanisms.
  • MTs have been found to protect against apoptosis/necrosis induced by oxidative stress, etoposide, cisplatin, doxorubicin and X-irradiation (Cai et al. (2004) Toxicol. Lett. 146, 217-226; Chimienti et al. (2001) Free Radicals Biol. Med. 31, 1179-1 190; Wang et al. (2001) J. Pharmacol. Exp. Ther. 298, 461-468).
  • Apo-MTs are effectively degraded by lysosomal cathepsins, while metal-conjugated MTs are more stable (Hahn et al. (2001) Exp. Mol. Med. 33, 32-36; Feldman et al. (1978) Biochim. Biophys. Acta 544, 638-646).
  • Baird et al (Biochem J. 2006 Feb 15;394(Pt l):275-83) proposes that MTs may stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment and Lee et al. (Glia. 2010 Aug;58(10): 1186-96) discloses that MT-3 may regulate lysosomal function in cultured astrocytes under both normal and oxidative conditions.
  • the MT transcript and protein described herein may be selected from, for example, metallothionein-lA (MT1A), metallothionein-lB (MT1B), metallothionein-lE (MT1E), metallothionein-lF (MT1F), metallothionein-lG (MT1G), metallothionein- 1H (MT1H), metallothionein-11 pseudogene (MTlIp or MTE), metallothionein-lL (MTIL or MTIR), metallothionem-lM (MTIM or MTIK), metallothionein-lX (MT1X), metallothionein-2 (MT2), metallothionein-2A (MT2A) metallothionein-3 (MT3) or metallothionein-4 (MT4).
  • MT1A metallothionein-lA
  • MT1B metallothionein-lE
  • MT1F metallothi
  • NP_005937.2 (MT1A); NP_005938.1 (MT1B); NP_783316.2 (MT1E);
  • NPJ 05940.1 (MT1F); NPJ)05941.1 (MT1G); NPJ)05942.1 (MT1H); NP_789846.1 (MTIM); NP_005943.1 (MT1X); NP_005944.1 (MT2); NP_005945.1 (MT3); and NP_116324.1 (MT4).
  • Further NCBI accession numbers for MT1 A, MT1E, MT2A and MTE-MT1IP are: NM_005946.2, NMJ 75617.3, NMJ)05953.3 and
  • lysosomal storage disorder refers to any of a group of diseases resulting from abnormal metabolism resulting in accumulation of a substrate (for example sulfatides, heparan sulphate, glycolipids, ceramide) in the lysosome.
  • a substrate for example sulfatides, heparan sulphate, glycolipids, ceramide
  • LSDs are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of lipids, glycoproteins (sugar-containing proteins) or so-called mucopolysaccharides.
  • the lysosome is commonly referred to as the cell's recycling center because it processes unwanted material into substances that the cell can utilize. Lysosomes break down this unwanted matter via enzymes, highly specialized proteins essential for survival. Lysosomal disorders are triggered when a particular enzyme exists in too small an amount or is missing altogether. When this happens, substances accumulate in the cell. In other words, when the lysosome doesn't function normally, excess products destined for breakdown and recycling are stored in the cell.
  • Hurler-Scheie disease a -L-lduronidase
  • LSDs include Activator Deficiency/GM2 Gangliosidosis, Alpha- mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease, Fabry disease, Farber disease, Fucosidosis, Galactosialidosis, Gaucher Disease, GM1 gangliosidosis, I-Cell disease/Mucolipidosis II, Infantile Free Sialic Acid Storage Disease/ISSD, Juvenile Hexosaminidase A Deficiency, Krabbe disease, Lysosomal acid lipase deficiency, Metachromatic Leukodystrophy, Mucopolysaccharidoses disorders, Multiple sulfatase deficiency, Niemann-Pick Disease, Neuronal Ceroid Lipofuscinoses,
  • Metachromatic Leukodystrophy is a demyelinating LSD due to mutations in the Arylsulfatase A (ARSA) gene and is a prototypical example of LSD with progressive accumulation of un-degraded substrates in the nervous system and secondary neuroinflammation and degeneration.
  • the genetic transmission of MLD is autosomal recessive and its overall incidence is estimated to be 1:40.000-1 :100.000 (von Figura and Jaeken (2001) Metachromatic Leukodystrophy.
  • the disease is classified according to the age at onset of symptoms into late infantile, with onset of symptoms before 2 years of age, juvenile - further subdivided in early and late, with symptoms onset before 16 years of age, and adult forms with a later onset.
  • Clinical manifestations consisting of severe and unrelenting motor and cognitive impairment, and disease progression are more severe in the early onset clinical variants, leading to death usually within the first decade of life.
  • a correlation between the phenotype of MLD patients and the type of ARSA mutation they bear has recently been demonstrated (Cesani et al. (2009) Hum Mutat, 30, E936-945; Biffi. et al. (2008) Clinical Genetics, 74, 349-357).
  • Nermann Pick disease is characterised by mutations in the SMPDl gene (disease types A and B) and mutations in NPCl and NPC2 (disease type C, NPC). Symptoms are related to the organs in which they accumulate. Enlargement of the liver and spleen (hepato splenomegaly) may cause reduced appetite, abdominal distension and pain as well as thrombocytopenia secondary to splenomegaly. NPC disease is an inherited disorder that is characterized by defects in intracellular cholesterol sorting and transport. Under normal conditions, the cholesteryl ester derived from low density lipoprotein mediates a complex feedback mechanism that stabilizes the intracellular concentration of cholesterol.
  • lipid trafficking In NPC patients, a defect in the activities of the NPCl and NPC2 proteins results in a very slow efflux of unesterified cholesterol.
  • the ratio of unesterified cholesterol and bis(monoacylglycerol)phosphate (BMP) is important within the internal lysosomal membrane for the efficient hydrolysis of membrane components to occur.
  • BMP bis(monoacylglycerol)phosphate
  • Sandhoff disease also knowrr as Jatzkewitz-Pilz syndrome and Hexosaminidase A and B deficiency, is a rare autosomal recessive, genetic, lipid storage disorder.
  • Sandhoff disease is associated with mutations in the HEXB gene which encodes Beta- hexosaminidase subunit beta.
  • the disease results from the inability to create the beta- hexosaminidase A and beta-hexosaminidase B, which is an enzyme that leads to a build-up of GM2 gangliosides in tissues of the body. This build-up is toxic at high levels, which leads to a progressive destruction of the central nervous system, damages the tissues and eventually leads to death.
  • Krabbe disease also Icnown as Globoid Cell Leukodystrophy or Galactosylceramide Lipidosis
  • Krabbe disease is caused by mutations in the GALC gene located on chromosome 14 (14q31), which causes a deficiency of galactocerebrosidase enzyme.
  • the build up of unmetabolized lipids affects the growth of the nerve's protective myelin sheath (the covering that insulates many nerves) and causes severe degeneration of motor skills.
  • MPS Mucopolysaccharidoses
  • MPS I is divided into three subtypes based on severity of symptoms. All three types result from an absence of, or insufficient levels of, the enzyme alpha-L-iduronidase.
  • MPS I H also called Hurler syndrome or a-L-iduronidase deficiency
  • MPS I S Scheie syndrome
  • MPS I H-S Hurler-Scheie syndrome
  • Hurler-Scheie syndrome is less severe than Hurler syndrome alone.
  • MPS II Hunter syndrome or iduronate sulfatase deficiency, is caused by lack of the enzyme iduronate sulfatase
  • MPS III Sanfilippo syndrome
  • Sanfilippo A is the most severe of the MPS III disorders and is caused by the missing or altered enzyme heparan N-sulfatase. Children with Sanfilippo A have the shortest survival rate among those with the MPS III disorders.
  • Sanfilippo B is caused by the missing or deficient enzyme alpha-N acetylglucosaminidase.
  • Sanfilippo C results from the missing or altered enzyme acetyl-CoAlpha-glucosaminide acetyltransferase.
  • Sanfilippo D is caused by the missing or deficient enzyme N-acetylglucosamine 6- sulfatase.
  • MPS IV Morquio syndrome
  • Type A N- acetylgalactosamine 6-sulfatase
  • Type B beta-galactosidase
  • MPS VI Maroteaux-Lamy syndrome, share many of the_physical symptoms found in Hurler syndrome and is casused be the deficient enzyme N-acetylgalactosamine 4- sulfatase.
  • MPS VII Sly syndrome, one of the least common forms of the mucopolysaccharidoses, is caused by deficiency of the enzyme beta-glucuronidase.
  • NCL Neuronal Ceroid Lipofuscinoses
  • lipopigments lipopigments
  • PPT1 palmitoyl protein thioesterase 1
  • the CLN2 gene encodes a 46kDa protein called lysosomal tripeptidyl peptidase I (TPPI) which cleaves tripeptides from terminal amine groups of partially unfolded proteins. Mutations of the CLN2 gene which encodes lysosomal tripeptidyl peptidase I (TPPI) typically result in a Late Infantile NCL (Jansky-Bielschowsky disease, LINCL) phenotype.
  • TPPI lysosomal tripeptidyl peptidase I
  • Gaucher' s disease is characterized by accumulation of the glycolipid glucocerebroside.
  • Three phenotypes have been described for Gaucher's disease that are denoted by the absence (type 1) or presence of neurological involvement during childhood (type 2) or adolescence (type 3) (Grabowski (1993) Adv Hum Genet; 21 :377-441).
  • Type 1 Gaucher's disease is panethnic, but is especially prevalent among persons of Ashkenazi Jewish descent.
  • the N370S and 84GG mutations are the most frequent mutations in the glucocerebrosidase gene among Ashkenazi Jews.
  • Type 1 Gaucher's disease tends to be severe and progressive in Japanese patients (see, Ida et al., Type 1 Gaucher Disease Patients: Phenotypic Expression and Natural History in Japanese Patients, Blood Cells, Molecules and Diseases, 1984, 24(5):73-81).
  • Type 3 Gaucher's disease associated with one or two copies of glucocerebrosidase gene variant L444P is prevalent in Swedish patients from the Norrbotten region.
  • an “antibody” is understood within the scope of the invention to refer to an antibody that is an intact molecule as well as fragments or portions thereof, such as Fab, F(ab')2, Fv and scFv.
  • sample is understood within the scope of the invention to refer to a sample which is derived from a subject.
  • the biological sample may be obtained directly from the subject or may be derived from cultured cells obtained from said subject.
  • the sample can be a whole blood sample, plasma sample, serum sample, urine or urinary sediment sample, broncheoalveolar lavage fluid sample, lymph sample, cerebrospinal fluid sample, saliva sample, semen sample, breast milk sample, or feces sample.
  • the test sample can also be a tissue sample from liver, kidney, muscle, heart, lung, spleen, lymph node, bone marrow, skin, blood vessels and valves, eye, or brain.
  • the test sample is a population of T lymphocytes or mononuclear cells.
  • polypeptide and “peptide” refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds.
  • protein encompasses proteins that have been modified e.g.
  • glycoproteins lipoproteins etc.
  • the term protein also encompasses homologues and derivatives of known proteins, and fragments thereof.
  • an “immunoassay” is understood within the scope of the invention to include an enzyme- linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), an enzyme immunoassay (EIA), an immunofluorescence assay (IF A) or a luminescence assay (LIA) or a Western Blot assay (WB) or any other immunoassay commonly known in the art.
  • ELISA enzyme- linked immunosorbant assay
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • IF A immunofluorescence assay
  • LIA luminescence assay
  • WB Western Blot assay
  • a “subject” refers to either a human or non-human animal.
  • non-human animals include vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), dogs, rodents (e.g., mice, rats, or guinea pigs), pigs and cats, etc.
  • the subject is a human.
  • FIG. 1 Metallothioneins are over-expressed in T lymphocytes of patients with MLD.
  • A 26 probes, including 5 metallothioneins (marked in gray), are differentially expressed in T lymphocytes of 24 patients with MLD compared to 24 age- and sex- matched controls (CTR) (fold change>1.5; False Discovery RateO.01).
  • CTR age- and sex- matched controls
  • each column represents an individual and each row a probe. As shown in the color bar, over-expression is visualized in red and under-expression is displayed in blue.
  • Figure 2 Metallothioneins-based expression signatures predict diagnosis and prognostic subtypes.
  • A) In the training set (n 32) individuals with MLD were classified with 94% sensitivity and 100% specificity.
  • (B) In the independent test set (n 16) patients with MLD were classified with 75% sensitivity and 100% specificity.
  • the confusion matrix indicates the number of individuals classified in each class using Prediction Analysis of Microarray (PAM).
  • C,D Classifying patients with MLD into clinically relevant prognostic classes based on gene expression.
  • Figure 3 Ingenuity Pathway Analysis shows association of oxidative stress- related pathways with MLD.
  • A Histogram showing the significance P value (- log(P value)) of the ten canonical pathways most significantly perturbed in MLD patients compared to controls based on Ingenuity Pathway Analysis. The pathways involved in oxidative stress (orange) and inflammation (blue) are highlighted.
  • B Graphical representation of the Protein Ubiquitination pathway, which is the most perturbed in MLD patients based on Ingenuity Pathway Analysis. The color scale indicates in gray the less differentially expressed genes and in red the more over- expressed ones.
  • MLD brain samples show diffuse architectural effacement, with medium-to-large size phagic cell engulfing the perivascular zones of the white matter.
  • Kluver-Barrera staining for myelin highlights diffuse dysmyelination in subcortical areas, with granular overload of phospholipidic components of myelin in the cytoplasm of the phagic cells.
  • FIG. 5 MTs are over-expressed in brain of LSD patients.
  • F, G Western blot immunoreactivity for MT proteins on samples from 5 LSD brains and 3 related controls (CTR), normalized for protein content as shown by actin immunoreactivity and compared to the signal from purified MT1 protein (CO+) (F), and on post-mortem CSF samples from 1 LSD patient, 1 Parkinson's Disease patient (PD) and 1 control individual (CTR) at decreasing volumes (25, 15 and 5 ⁇ ) (G).
  • MTs are a dynamic marker of disease progression and response to treatment
  • Figure 7 MT protein abundance in brain of GLD mice.
  • Figure 8 MT role is investigated in in vitro cultures.
  • A relative MT1 and MT2 mRNA expression in pure mouse astrocyte cultures after 6 (dark gray bars) and 24 (light gray bars) hours of contact with conditioned medium from resting or LPS- activated microglia, as compared to LPS-treated control astrocytes.
  • PBMC Peripheral blood mononuclear cells
  • CSF Post-mortem cerebrospinal fluid
  • mice were sacrificed at a late, symptomatic stage of the disease (10 months for MLD and MPS I, 35 days for GLD and MPS III and 3 to 4 months for SD); for the GLD model, mice were sacrificed also at day 9-12, and at day 40 after having received lethal irradiation and total bone marrow transplantation from wild-type sibling donors at post-natal day 8 as described (Visigalli et al. (2009) Neurobiology of disease, 34, 51-62). After sacrifice, blood was collected in EDTA and peripheral cells were obtained through lysis and stored at -80°C, while brains were perfused with PBS, collected and stored at -80°C.
  • RIN RNA integrity number
  • RNA pellet was washed once with 70% ethanol, let dry and re-suspended in RNase-free water.
  • RNA from mouse peripheral blood cells and astrocyte co-cultures was extracted respectively with RNeasy plus Micro and Mini Kits (Qiagen).
  • RNA samples 350 ng of total RNA from each specimen were transcribed into biotinilated cRNA (TotalPrep RNA amplification kit - Ambion). cRNA was purified and its concentration and integrity were determined (2100 Bioanalyzer - Agilent).
  • Microarray analysis was performed according to the MIAME guidelines. The data discussed in this application have been deposited in NCBI's Gene Expression Omnibus (Edgar et al. (2002) Nucleic acids research, 30, 207-210) and are accessible through GEO Series accession number GSE23350
  • IP A Ingenuity Pathway Analysis
  • Receiver-operating characteristic (ROC) curve was obtained plotting the average ⁇ SEM of False Positive and False Negative fractions for MT1A, MT1E, MT2A and MTE qPCR data, as calculated using the Web-based Calculator for ROC Curves at http://www.rad.jlrmi.edu/jeng/iavarad/roc/JROCFITi.html. Area under the ROC curve was calculated using the trapezoid rule. Primary cultures of pure astrocytes
  • cortical astrocytes Primary cultures of cortical astrocytes were prepared from 2 to 3 day-old C57 mice according to Consonni et al. (2011) Mol Cell Neurosci, 48, 151-160. Briefly, after dissection, cortices were cut into small sections with a razor blade. The pieces were collected and washed twice in Hank's Balanced Salt Solution supplemented with 10 mM Hepes Na pH 7.4, 12 mM MgS04, 50 U/ml penicillin and 50 ⁇ g/ml streptomycin (Gibco).
  • the tissue was then incubated, in two subsequent steps, with 2.5 mg/ml trypsin type IX in presence of 1 mg/ml deoxyribonuclease (Calbiochem) for 10 min at 37 °C and mechanically dissociated.
  • the supernatant obtained was diluted 1 :1 in medium containing 10% donor horse serum (PAA Laboratories GmbH). After centrifugation (100 g for 10 min), cells were plated in Minimum Essential Medium Eagle supplemented with 10% donor horse serum, 33 mM glucose, 2 mM glutamax (Gibco), 50 U/ml penicillin, 50 ⁇ g/ml streptomycin.
  • astrocytes were trypsinized and replated in Minimum Essential Medium Eagle supplemented with 10% donor horse serum, 33 mM glucose, 2 mM glutamax (Gibco), 50 U/ml penicillin, 50 ⁇ g/ml streptomycin onto poly-lysine-coated plastic multiwells. Cultures were maintained at 37°C in a 5% C0 2 humidified incubator, and experiments were performed within 3 days after re-plating. Conditioned medium obtained from resting or activated (with 10 ng/ml LPS) microglia was administered to pure astrocytic cultures in substitution of their culture medium either 6 or 24 hours before RNA extraction. 10 ng/ml LPS was added directly to astrocyte culture medium either 6 or 24 hours before RNA extraction and used as negative control for activation.
  • Minimum Essential Medium Eagle supplemented with 10% donor horse serum, 33 mM glucose, 2 mM glutamax (Gibco), 50 U/ml penicillin, 50 ⁇ g/ml streptomycin onto
  • Serum- containing medium was added in order to inactivate papain and a first centrifugation was done at 1000 rpm for 10 minutes. After removing supernatant, this step was repeated with fresh serum-containing medium. After the second centrifugation, the pellet was more finely dissociated with a 200 ⁇ tip pipette and then centrifuged at 1000 rpm for 10 minutes. After resuspension of the pellet, the cell suspension was plated into a 12-multiwell plate (usually 10 wells/hemisphere, corresponding roughly to 40 cm ) in Minimum Essential Medium Eagle supplemented with 10% donor horse serum, 33 mM glucose, 2 mM glutamax (Gibco), 50 U/ml penicillin, 50 ⁇ g/ml streptomycin.
  • Minimum Essential Medium Eagle supplemented with 10% donor horse serum, 33 mM glucose, 2 mM glutamax (Gibco), 50 U/ml penicillin, 50 ⁇ g/ml streptomycin.
  • a 24h-incubation was performed for: (i) 10 ⁇ dexamethasone; (ii) 10% serum from wild-type and late-symptomatic GLD mice; (iii) MT1 and MT2, 20 ⁇ g/ml each. At the end of stimulation, cells were collected for RNA extraction as previously described.
  • RNA from human T lymphocytes and brains and from murine brains and cells were reverse-transcribed into cDNA (High Capacity cDNA Reverse Transcription Kit and Superscript III First Strand Synthesis - Life Technologies).
  • qPCR was performed on an ABI 7900HT or Viia7 (Life Technologies) and analyzed " by comparative threshold cycle method using Taqman Gene Expression Assay primers and probes for human Metallothionein 1A, IE, 2 A and E, murine Metallothionein 1 and 2 and CXCL11, and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) as reference gene (Life Technologies).
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • Immunohistochemistry Consecutive 4- ⁇ thick sections from formalin-fixed paraffin-embedded brain tissue samples of MLD and controls brains were prepared and stained with routine Hematoxylin-Eosin and Kluver-Barrera (Luxol Fast Blue) method for myelin. Irrimunostaining with anti-metallothionein (MT) monoclonal antibodies (Dako, clone E9, diluition 1 :50) was performed on both MLD and control brain sections.
  • MT anti-metallothionein
  • the membrane was stripped for 15' with Restore Buffer (Thermo Scientific), blocked and incubated for 3h with goat -actin antibody (Santa Cruz Biotechnology) 1 : 10000 in TBS-T 3% milk and lh with a-goat HRP-conjugated antibody (Santa Cruz Biotechnology) 1 :20000 in TBS-T 3% milk. Blots were developed with ECL system (Millipore).
  • Example 2 - Metallothioneins are over-expressed in T lymphocytes of patients with MLD
  • MT1A, MT1E, MT2A and MTE- MT1IP include a cluster comprising four members of the family of metallothioneins (MT1A, MT1E, MT2A and MTE- MT1IP; NCBI RefSeq: NM_005946.2, NM_175617.3, NM 005953.3 and NR_003669.1 respectively) and the metallothionein pseudogene MT1P3- C20ORF127, which show on average a 1.60-fold over-expression in MLD patients as compared to controls.
  • differentially expressed genes were related to immune function (such as TIMD4, TNFSF13B, GNLY or GZMH).
  • Example 3 Gene expression signatures predict diagnosis and prognostic subtypes of MLD
  • To identify a transcriptional profile associated with MLD we randomly chose a subset of patient and control samples to build first a diagnostic and then a prognostic classifier for MLD.
  • This training set comprised -67% of the subjects (32 of 48 individuals), including 8 randomly selected early-onset MLD patients, 8 randomly selected late-onset MLD patients, and 16 age-matched controls.
  • Biomarkers useful for predicting disease prognosis are clinically needed for MLD.
  • a second classifier specifically designed to predict clinical prognostic subtypes of MLD based on gene expression changes in T lymphocytes.
  • PAM Proc Natl Acad Sci U S A, 99, 6567-6572
  • 31-gene prognostic classifier that again included MTl A, MTIE and MT2A as well as most of the 15 genes comprising the diagnostic classifier (Table 3).
  • the 31-gene prognostic classifier accurately classified individuals into the prognostic groups of early-onset or late-onset disease, and controls.
  • Example 4 Pathway analysis indicates a role for metallothioneins in the MLD disease process
  • IP A Ingenuity Pathway Analysis
  • NRF2-mediated pathway is implicated in the recruitment of antioxidant protems aimed at reducing the cellular oxidative damage, and in the repair and removal of damaged proteins from the cytoplasm through activation of chaperones and stress response proteins, together with ubiquitination and proteasomal degradation proteins.
  • Protein Ubiquitination pathway is the most perturbed in MLD patients based on Ingenuity Pathway Analysis. Protein Ubiquitination pathway genes that were less differentially expressed genes and more over-expressed were identified ( Figure 3B).
  • Example 5 - Metallothioneins arc over-expressed in the brain of patients with MLD
  • Example 6 Metallothioneins are general markers of nervous tissue damage in LSDs
  • mice affected by MLD, GLD, MPSI and III and SD, all characterized by demyelination and/or neurodegeneration of differential severity.
  • Both murine MT1 and MT2, orthologous of the human isoforms MT1 and MT2 were significantly over- expressed in LSD mouse models ( Figure 7A).
  • Figure 7A As observed in humans, the extent of expression changes varied in each specific lysosomal storage disease, with the mouse model for GLD showing the highest transcript levels, (fold change of 5.5 for MT1 and of 8.0 for MT2).
  • Example 7 - Metallothioneins are dynamic markers of disease progression and therapeutic response.
  • Hematopoietic stem cell transplantation from healthy donors has been introduced historically in many LSDs and more recently in GLD as a procedure capable of delaying disease onset and attenuating the disease phenotype by providing the functional enzyme to the affected nervous tissue by the means of CNS-infiltrating donor-derived cells (Escolar et al (2005), N. Engl. J. Med., 352, 2069-2081).
  • GLD animals at post-natal day 8 underwent bone marrow transplantation from wild-type donors. Transplanted mice were sacrificed at day 40 when only mildly symptomatic.
  • MT expression levels in treated animals were significantly lower than the levels measured in untreated age-matched affected controls, which were moribund at this disease stage, indicating that MTs could represent a marker not only of disease progression but also of response to treatment (Figure 6A).
  • Example 8 The role of metallothioneins in brain oxidative stress and inflammation response is dissected in vitro.
  • microglia are known to be activated in the neuroiiiflammatory and neurodegenerative context typical of LSDs (Visigalli et al (2009)while astrocytes are the main source of MTs in the affected brain (Chung et al (2004) Journal of neurochemistry, 88, 454-461), as also confirmed by our data on mouse and human samples.
  • LSDs involve the CNS, and the LSD-associated neurological manifestations represent a major cause of morbidity and mortality for the affected patients.
  • a more reachable source such as blood cells
  • the transcriptome analysis performed on T lymphocytes of patients provided a progression biomarker useful not only for MLD but also for the broader class of LSDs with CNS involvement.
  • MT expression could possibly be activated as a consequence of the block of autophagy, which has been shown to be a direct consequence of the lysosomal dysfunction (Settieri et al (2008) Hum Mol Genet, 17, 119-129; Baird et al (2006) Biochem J, 394, 275-283), as an attempt by the cell to lower the oxidative stress, counteract the accumulating pro-apoptotic stimuli and mitigate tissue inflammation.
  • analysis on microarray data points to a selective perturbation of pathways related to oxidative stress and inflammation in MLD patients' samples.
  • MT increase both in the CNS and in blood, could also reflect a cell-extrinsic response to still unidentified soluble factors, as suggested by the mechanistic studies performed with GLD mouse serum.
  • Table 1 List of genes most differentially expressed in MLD patients compared to age- and sex-matched controls.
  • ATP1B1 ATPase Na+/K+ transporting, beta 1 polypeptide 0.6482 0.000070 0
  • TNFSF13B TNF (ligand) superfamily member 13b 1.5734 0.000556 0
  • TNFSF13B TNF (ligand) superfamily member 13b 1.5327 0.001947 0
  • EMP1 epithelial membrane protein 1 1.5823 0.002258 0
  • PRDM1 PR domain containing 1 with ZNF domain 1.5055 0.003636 0
  • GZMH granzyme H (cathepsin G-like 2, protein h-CCPX) 1.9466 0.008254 0
  • Table 2 List of genes belonging to the binary diagnostic classifier used to discriminate between MLD patients and controls.
  • ALOX5AP Arachidonate 5-lipoxygenase-activating protein 0.1316 -0.1316
  • IFITM2 Interferon induced transmembrane protein 2 -0.0262 0.0262
  • IFITM1 Interferon induced transmembrane protein 1 -0.0244 0.0244
  • Table 3 List of genes belonging to the diagnostic classifier used to discriminate between early-onset MLD patients, late-onset MLD patients and controls.
  • CNN2 Calponin 2 0 -0.1216 0
  • ALOX5AP Arachidonate 5-lipoxygenase-activating protein 0 0 -0.0994
  • SH2D1A SH2 domain protein 1A, Duncan's disease 0.0368 0 0
  • ANAPC10 anaphase promoting complex subunit 0 1.190 1.90E- -03
  • HLA-A major histocompatibility complex class I, A 1.033 6.01 E- -01
  • HLA-B major histocompatibility complex class I, B 1.042 4.95E- -01
  • HSP90AA1 heat shock protein 90kDa alpha (cytosolic), class A member 1 1.033 7.53E- -01
  • HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) 1.107 4.28E- -02
  • HSPA6 heat shock 70kDa protein 6 (HSP70B') 1122 3.85E- -01
  • PSMA4 proteasome (prosome, macropain) subunit alpha type, 4 1.105 8.02E- -02
  • PSMA5 proteasome (prosome, macropain) subunit alpha type, 5 1.074 7.85E- -02
  • PSMB1 proteasome (prosome, macropain) subunit beta type, 1 1.035 4.01 E- -01
  • PSMB4 proteasome (prosome, macropain) subunit beta type, 4 1.024 4.61 E- -01
  • PSMB6 proteasome (prosome, macropain) subunit beta type, 6 1.058 2.70E- -01
  • PSMB8 proteasome prosome, macropain subunit, beta type, 8 (large 1.056 4.75E- -01 multifunctional peptidase 7)
  • PSMB9 proteasome prosome, macropain subunit, beta type, 9 (large 1.078 2.48E- -01 multifunctional peptidase 2)
  • PSMC1 proteasome prosome, macropain
  • 26S subunit ATPase
  • PSMC2 proteasome prosome, macropain
  • 26S subunit ATPase
  • PSMC4 proteasome prosome, macropain
  • 26S subunit ATPase
  • PSMC6 proteasome prosome, macropain
  • 26S subunit ATPase
  • PSMD6 proteasome prosome, macropain
  • 26S subunit non-ATPase
  • PSME1 proteasome (prosome, macropain) activator subunit 1 PA28 1.030 5.81 E- -01 alpha
  • PSME2 proteasome (prosome, macropain) activator subunit 2 PA28 1.040 5.84E- -01 beta
  • TAP1 transporter 1 1, ATP-binding cassette, sub-family B (MDR/TAP) 1.051 3.59E- -01
  • TAP2 transporter 2 ATP-binding cassette, sub-family B (MDR/TAP) 1.174 1.42E- -02
  • UBE2E1 ubiquitin-conjugating enzyme E2E 1 (UBC4/5 homoiog, yeast) 1.063 3.09E- -01
  • UBE2J1 ubiquitin-conjugating enzyme E2, J1 (UBC6 homoiog, yeast) 1.120 6.47E- -02
  • UBE2N ubiquitin-conjugating enzyme E2N (UBC13 homoiog, yeast) 1.048 2.13E- -01
  • UBE4A ubiquitination factor E4A (UFD2 homoiog, yeast) 1.052 4.43E- -01
  • UBE4B ubiquitination factor E4B (UFD2 homoiog, yeast) 1.080 8.18E- -02
  • UCHL3 ubiquitin carboxyl-terminal esterase L3 ubiquitin thiolesterase 1.070 1.35E- -01
  • NDUFAB1 NADH dehydrogenase (ubiquinone) 1 alpha/beta subcomplex, 1.140 5.07E- -02
  • NDUFC1 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1 , 1.087 9.10E- -02
  • PPA1 pyrophosphatase (inorganic) 1 1.048 3.51 E- -01
  • SDHC succinate dehydrogenase complex subunit C, integral 1.016 6.96E- -01 membrane protein, 15kDa
  • AIFM1 apoptosis-inducing factor, mitochondrion-associated, 1 1.032 3.75E- -01
  • APH1A anterior pharynx defective 1 homolog A (C. elegans) 1.015 8.34E- -01
  • APH1 B anterior pharynx defective 1 homolog B (C. elegans) 1.029 4.24E- -01
  • FIS1 fission 1 mitochondria outer membrane homolog (S. 1.006 9.02E- -01 cerevisiae)
  • MAP2K4 mitogen-activated protein kinase kinase 4 1.008 8.85E- -01
  • MAPK9 mitogen-activated protein kinase 9 1.028 6.41 E- -01
  • NDUFAB1 NADH dehydrogenase (ubiquinone) 1 alpha/beta subcomplex, 1.140 5.07E- -02 1 , 8kDa
  • SDHC succinate dehydrogenase complex subunit C, integral 1.016 6.96E- -01 membrane protein, 15kDa
  • CD3D CD3d molecule delta (CD3-TCR complex) 1.101 6.06E- -02
  • ELK1 ELK1 member of ETS oncogene family 1.006 9.34E- -01
  • EP300 E1 A binding protein p300 1.054 4.08E- -01
  • GTF2A2 general transcription factor IIA, 2, 12kDa 1.092 2.24E- -01
  • GTF2B general transcription factor IIB 1.037 3.44E- -01
  • GTF2E1 general transcription factor HE, polypeptide 1 , alpha 56kDa 1.077 5.60E- -02
  • GTF2E2 general transcription factor HE, polypeptide 2, beta 34kDa 1.017 6.26E- -01
  • GTF2F2 general transcription factor IIF polypeptide 2, 30kDa 1.083 7.47E- -02
  • GTF2H1 general transcription factor IIH, polypeptide 1 , 62kDa 1.145 2.76E- -02
  • GTF2H5 general transcription factor IIH polypeptide 5 1.185 1.55E- -02
  • HMGB1 high-mobility group box 1 1.198 1.35E- -02
  • HSP90AA1 heat shock protein 90kDa alpha (cytosolic), class A member 1 1.033 7.53E- -01
  • HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) 1.107 4.28E- -02
  • HSPA6 heat shock 70kDa protein 6 (HSP70B') 1.122 3.85E- -01
  • IL5 interleukin 5 colony-stimulating factor, eosinophil
  • MAP2K1 mitogen-activated protein kinase kinase 1 1.005 9.21 E- -01
  • MAP2K4 mitogen-activated protein kinase kinase 4 1.008 8.85E- -01
  • MAP3K1 mitogen-activated protein kinase kinase kinase 1 1.136 1.72E- -01
  • MAP3K7 mitogen-activated protein kinase kinase kinase 7 1.035 4.72E- -01
  • MAPK1 mitogen-activated protein kinase 1 1.042 4.45E- -01
  • APK9 mitogen-activated protein kinase 9 1.028 6.41 E- -01
  • MAPK14 mitogen-activated protein kinase 14 1.006 8.97E- -01
  • NCOA1 nuclear receptor coactivator 1 1.051 3.70E- -01
  • NCOA2 nuclear receptor coactivator 2 1.010 8.62E- -01
  • NCOA3 nuclear receptor coactivator 3 1.030 5.51 E- -01
  • NFAT5 nuclear factor of activated T-cells 5 tonicity-responsive 1.056 4.46E- -01
  • NR3C1 nuclear receptor subfamily 3 group C, member 1 1.017 7.32E- -01
  • NRIP1 nuclear receptor interacting protein 1 1.007 9.20E- -01
  • PIK3CA phosphoinositide-3-kinase catalytic, alpha polypeptide 1.040 3.60E- -01
  • PLAU plasminogen activator urokinase 1.273 3.78E- -02
  • RNA II DNA directed polypeptide A, 220kDa 1.011 8.32E- -01
  • RNA II DNA directed polypeptide C, 33kDa 1.005 9.29E- -01
  • RNA II DNA directed polypeptide D 1.040 2.99E- -01
  • RNA II DNA directed polypeptide F 1.056 3.20E- -01
  • RNA II DNA directed polypeptide G 1.066 2.79E- -01
  • RNA II DNA directed polypeptide H 1.027 5.75E- -01
  • RNA II DNA directed polypeptide I, 14.5kDa 1.000 9.96E- -01
  • RNA polymerase II (DNA directed) polypeptide J, 13.3kDa 1.062 3.99E- -01 polymerase (RNA) II (DNA directed) polypeptide J2 1.035 7.06E- -01
  • PPP3CA protein phosphatase 3, catalytic subunit, alpha isozyme 1.017 7.37E- -01
  • PTGS2 prostaglandin-endoperoxide synthase 2 prostaglandin G/H 1.150 3.14E- -01 synthase and cyclooxygenase
  • TAF2 TAF2 RNA polymerase II TAF2 TAF2 RNA polymerase II
  • TATA box binding protein (TBP)- 1.051 3.70E- -01 associated factor TAF2 TAF2 RNA polymerase II
  • TATA box binding protein (TBP)- 1.051 3.70E- -01 associated factor TAF2 TAF2 RNA polymerase II
  • TATA box binding protein (TBP)- 1.051 3.70E- -01 associated factor 150kDa
  • TAF5 TAF5 RNA polymerase II TAF5 TAF5 RNA polymerase II
  • TAF9 TAF9 RNA polymerase II TATA box binding protein (TBP)- 1.116 1.27E- -01 associated factor, 32kDa
  • TAF10 TAF10 RNA polymerase II TATA box binding protein (TBP)- 1.007 8.82E- -01 associated factor, 30kDa
  • TAF12 TAF12 RNA polymerase II TATA box binding protein (TBP)- 1.073 1.05E- -01 associated factor, 20kDa
  • TAF13 TAF13 RNA polymerase II TATA box binding protein (TBP)- 1.033 6.37E- -01 associated factor, 18kDa
  • TAF15 TAF15 RNA polymerase II TAF15 TAF15 RNA polymerase II
  • TATA box binding protein (TBP)- 1.064 1.93E- -01 associated factor TAF15 TAF15 RNA polymerase II
  • TATA box binding protein (TBP)- 1.064 1.93E- -01 associated factor TAF15 TAF15 RNA polymerase II
  • TATA box binding protein (TBP)- 1.064 1.93E- -01 associated factor 68kDa
  • TAF1 L TAF1 RNA polymerase II
  • TAF4B TAF4b RNA polymerase II TAF4B TAF4b RNA polymerase II
  • TATA box binding protein (TBP) TATA box binding protein- 1.036 4.90E- -01 associated factor, 105kDa
  • TAF9B TAF9B RNA polymerase II TAF9B TAF9B RNA polymerase II
  • CD74 CD74 molecule major histocompatibility complex, class II 1.1 13 2.19E-01 invariant chain
  • HLA-A major histocompatibility complex class I, A 1.033 6.01 E-01
  • HLA-B major histocompatibility complex class I, B 1.042 4.95E-01
  • HLA-DMB major histocompatibility complex class II, DM beta 1.079 4.13E-01
  • HLA-DOA major histocompatibility complex class II, DO alpha 1.083 2.97E-01
  • HLA-DPA1 major histocompatibility complex, class II, DP alpha 1 1.193 2.41 E-01
  • HLA-DQA1 major histocompatibility complex, class II, DQ alpha 1 1.204 2.98E-01
  • HLA-F major histocompatibility complex class I, F 1.148 1.27E-01
  • HLA-G major histocompatibility complex class I, G 1.055 6.14E-01
  • PSMB8 proteasome prosome, macropain subunit, beta type, 8 (large 1.056 4.75E-01 multifunctional peptidase 7)
  • PSMB9 proteasome prosome, macropain subunit, beta type, 9 (large 1.078 2.48E-01 multifunctional peptidase 2)
  • TAP1 transporter 1 ATP-binding cassette, sub-family B (MDR/TAP) 1.051 3.59E-01-
  • TAP2 transporter 2 ATP-binding cassette, sub-family B (MDR/TAP) 1.174 1.42 E-02
  • HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) 1.107 4.28E-02
  • MPP6 membrane protein, palmitoylated 6 (MAGUK p55 subfamily 1.047 4.61 E-01 member 6)
  • NME7 non-metastatic cells 7, protein expressed in (nucleoside- 1.062 2.42E-01 diphosphate kinase)
  • PNPT1 polyribonucleotide nucleotidyltransferase 1 1.12 3.82E-02
  • POLE2 polymerase DNA directed
  • epsilon 2 p59 subunit
  • RNA II DNA directed polypeptide A, 220kDa 1.011 8.32E-01
  • RNA II DNA directed polypeptide C, 33kDa 1.005 9.29E-01
  • RNA II DNA directed polypeptide F 1.056 3.20E-01
  • RNA II DNA directed polypeptide G 1.066 2.79E-01
  • RNA II DNA directed polypeptide H 1.027 5.75E-01
  • RNA II DNA directed polypeptide J, 13.3kDa 1.062 3.99E-01
  • RNA III DNA directed polypeptide A, 155kDa 1.011 8.72E-01
  • RNA III DNA directed polypeptide B 1.037 5.06E-01
  • RNA III DNA directed polypeptide C (62kD) 1.068 1.05E-01
  • RNA III DNA directed polypeptide H (22.9kD) 1.036 3.59E-01
  • RNA III DNA directed polypeptide K, 12.3 kDa 1.105 1.14E-01
  • PRIM1 primase DNA, polypeptide 1 (49kDa) 1.056 3.94E-01
  • PRPS2 phosphoribosyl pyrophosphate synthetase 2 1.16 4.62E-03
  • PSMC1 proteasome proteasome (prosome, macropain) 26S subunit, ATPase, 1 1.052 2.11 E-01
  • PSMC2 proteasome prosome, macropain
  • 26S subunit ATPase
  • PSMC4 proteasome prosome, macropain 26S subunit, ATPase, 4 1.04 3.05E-01
  • PSMC6 proteasome prosome, macropain 26S subunit, ATPase, 6 1.051 2.44E-01
  • TAF9 TAF9 RNA polymerase II TATA box binding protein (TBP)- 1.116 1.27E-01 associated factor, 32kDa
  • VPS4B vacuolar protein sorting 4 homolog B (S. cerevisiae) 1.026 6.24E-01
  • GTF2A2 general transcription factor IIA, 2, 12kDa 1.092 2.24E-01
  • GTF2B general transcription factor IIB 1.037 3.44E-01
  • GTF2E1 general transcription factor HE, polypeptide 1 , alpha 56kDa 1.077 5.60E-02
  • GTF2E2 general transcription factor HE, polypeptide 2, beta 34kDa 1.017 6.26E-01
  • GTF2H1 general transcription factor IIH, polypeptide 1 , 62kDa 1.145 2.76E-02
  • GTF2H5 general transcription factor IIH, polypeptide 5 1.185 1.55E-02
  • RNA II DNA directed polypeptide C, 33kDa 1 .005 9.29E-01
  • RNA II DNA directed polypeptide F 1.056 3.20E-01
  • RNA II DNA directed polypeptide G 1.066 2.79E-01
  • RNA II DNA directed polypeptide H 1.027 5.75E-01
  • RNA II DNA directed polypeptide J, 13.3kDa 1.062 3.99E-01
  • RNA II DNA directed polypeptide J2 1.035 7.06E-01
  • TAF2 TAF2 RNA polymerase II TAF2 TAF2 RNA polymerase II
  • TATA box binding protein (TBP)- 1.051 3.70E-01 associated factor TAF2 TAF2 RNA polymerase II
  • TATA box binding protein (TBP)- 1.051 3.70E-01 associated factor 150kDa
  • TAF5 TAF5 RNA polymerase II TAF5 TAF5 RNA polymerase II
  • TAF9 TAF9 RNA polymerase II TATA box binding protein (TBP)- 1.116 1.27E-01 associated factor, 32kDa
  • TAF12 TAF12 RNA polymerase II TAF12 TAF12 RNA polymerase II
  • TATA box binding protein (TBP)- 1.073 1.05E-01 associated factor 20kDa
  • TAF13 TAF13 RNA polymerase II TATA box binding protein (TBP)- 1.033 6.37E-01 associated factor, 18kDa
  • TAF15 TAF15 RNA polymerase II TAF15 TAF15 RNA polymerase II
  • TATA box binding protein (TBP)- 1.064 1.93E-01 associated factor TAF15 TAF15 RNA polymerase II
  • TATA box binding protein (TBP)- 1.064 1.93E-01 associated factor TAF15 TAF15 RNA polymerase II
  • TATA box binding protein (TBP)- 1.064 1.93E-01 associated factor TAF15 TAF15 RNA polymerase II
  • TAF10 TAF10 RNA polymerase II TATA box binding protein (TBP)- 1.007 8.82E-01
  • TAF1 L TAF1 RNA polymerase II
  • TAF4B TAF4b RNA polymerase II TAF4B TAF4b RNA polymerase II
  • TATA box binding protein (TBP)- 1.036 4.90E-01 associated factor TAF4B TAF4b RNA polymerase II
  • TATA box binding protein (TBP)- 1.036 4.90E-01 associated factor TAF4B TAF4b RNA polymerase II
  • TATA box binding protein (TBP)- 1.036 4.90E-01 associated factor 105kDa
  • TAF9B TAF9B RNA polymerase II TAF9B TAF9B RNA polymerase II
  • APAF1 apoptotic peptidase activating factor 1 1.036 4.74E-01
  • CD3D CD3d molecule CD3D CD3d molecule, delta (CD3-TCR complex) 1.101 6.06E-02
  • CD3G CD3g molecule CD3G CD3g molecule, gamma (CD3-TCR complex) 1.016 7.89E-01
  • GZMB granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated 1.389 5.20E-03 serine esterase 1 )
  • HLA-A major histocompatibility complex class I, A 1.033 6.01 E-01
  • HLA-B major histocompatibility complex class I, B 1.042 4.95E-01
  • HLA-DMB major histocompatibility complex class II, DM beta 1.079 4.13E-01
  • HLA-DOA major histocompatibility complex class II, DO alpha 1.083 2.97E-01
  • HLA-DPAT major histocompatibility complex class II, DP alpha 1 1.193 2.41 E-01
  • HLA-DQA1 major histocompatibility complex, class II, DQ alpha 1 1.204 2.98E-01
  • HLA-F major histocompatibility complex class I, F 1.148 1.27E-01
  • HLA-G major histocompatibility complex class I, G 1.055 6.14E-01
  • CTBP1 C-terminal binding protein 1 1.007 8.92E-01
  • GTF2E1 general transcription factor HE, polypeptide 1 , alpha 56kDa 1.077 5.60E-02
  • GTF2F2 general transcription factor IIF polypeptide 2, 30kQa 1.083 7.47E-02
  • GTF2H1 general transcription factor IIH, polypeptide 1 , 62kDa 1.145 2.76E-02
  • GTF2H5 general transcription factor IIH, polypeptide 5 1.185 1.55E-02
  • HNRNPD heterogeneous nuclear ribonucleoprotein D (AU-rich element 1.015 7.86E-01
  • RNA binding protein 1 37kDa
  • MAP2K1 mitogen -activated protein kinase kinase 1 1.005 9.21 E-01
  • NCOA1 nuclear receptor coactivator 1 1.051 3.70E-01
  • NR3C1 nuclear receptor subfamily 3 group C, member 1 (glucocorticoid 1.017 7.32E-01 receptor)
  • NRIP1 nuclear receptor interacting protein 1 1.007 9.20E-01
  • RNA II DNA directed polypeptide C, 33kDa 1.005 9.29E-01
  • RNA II DNA directed polypeptide F 1.056 3.20E-01
  • RNA II DNA directed polypeptide G 1.066 2.79E-01
  • RNA II DNA directed polypeptide H 1.027 5.75E-01
  • RNA POLR2J polymerase II (DNA directed) polypeptide J, 13.3kDa 1.062 3.99E-01
  • RNA POLR2J2/P polymerase II (DNA directed) polypeptide J2 1.035 7.06E-01
  • PRKDC protein kinase DNA-activated, catalytic polypeptide 1.025 - • 5-.91 E-01
  • TAF2 TAF2 RNA polymerase II TAF2 TAF2 RNA polymerase II
  • TATA box binding protein (TBP)- 1.051 3.70E-01 associated factor TAF2 TAF2 RNA polymerase II
  • TATA box binding protein (TBP)- 1.051 3.70E-01 associated factor 150kDa
  • TAF5 TAF5 RNA polymerase II TAF5 TAF5 RNA polymerase II
  • TAF9 TAF9 RNA polymerase II TATA box binding protein (TBP)- 1.116 1.27E-01 associated factor, 32kDa
  • TAF12 TAF12 RNA polymerase II TAF12 TAF12 RNA polymerase II
  • TATA box binding protein (TBP)- 1.073 1.05E-01 associated factor 20kDa
  • TAF13 TAF13 RNA polymerase II TATA box binding protein (TBP)- 1.033 6.37E-01 associated factor, 18kDa
  • TAF15 TAF15 RNA polymerase II TAF15 TAF15 RNA polymerase II
  • TATA box binding protein (TBP)- 1.064 1.93E-01 associated factor TAF15 TAF15 RNA polymerase II
  • TATA box binding protein (TBP)- 1.064 1.93E-01 associated factor TAF15 TAF15 RNA polymerase II
  • TATA box binding protein (TBP)- 1.064 1.93E-01 associated factor TAF15 TAF15 RNA polymerase II
  • TAF10 TAF10 RNA polymerase II TATA box binding protein (TBP)- 1.007 8.82E-01
  • TAF1 L TAF1 RNA polymerase II
  • TAF4B TAF4b RNA polymerase II TAF4B TAF4b RNA polymerase II
  • TATA box binding protein (TBP)- 1.036 4.90E-01 associated factor TAF4B TAF4b RNA polymerase II
  • TATA box binding protein (TBP)- 1.036 4.90E-01 associated factor TAF4B TAF4b RNA polymerase II
  • TATA box binding protein (TBP)- 1.036 4.90E-01 associated factor 105kDa
  • TAF9B TAF9B RNA polymerase II TAF9B TAF9B RNA polymerase II
  • AKR1A1 aldo-keto reductase family 1 member A1 (aldehyde reductase) 1.009 8.80E- -01
  • EIF2AK3 eukaryotic translation initiation factor 2-alpha kinase 3 1.047 3.23E- -01
  • HERPUD1 homocysteine-inducible, endoplasmic reticulum stress- 1.092 7.29E- -02 inducible, ubiquitin-like domain member 1
  • HMOX1 heme oxygenase (decycling) 1 1.057 6.24E- -01

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Abstract

L'invention concerne une méthode de diagnostic ou de pronostic d'un trouble du stockage lysosomal chez un sujet, cette méthode consistant à (i) se procurer un échantillon provenant dudit sujet, (ii) déterminer le niveau d'expression d'une métallothionéine dans ledit échantillon, (iii) comparer le niveau d'expression de la métallothionéine dans ledit échantillon avec le niveau d'expression de la métallothionéine dans un échantillon provenant de sujets sains ou dans un échantillon antérieur du même sujet.
PCT/IB2012/054469 2011-08-30 2012-08-30 Biomarqueurs de troubles du stockage lysosomal WO2013030785A1 (fr)

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GB201114909A GB201114909D0 (en) 2011-08-30 2011-08-30 Biomarkers for lysosomal storage disorders
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WO2018136435A1 (fr) * 2017-01-17 2018-07-26 Children's Medical Center Corporation Compositions et procédés de traitement de maladies et de troubles du stockage lysosomial
WO2018136434A1 (fr) * 2017-01-17 2018-07-26 Children's Medical Center Corporation Compositions et méthodes de diagnostic et de traitement des maladies peroxysomales
WO2020123511A3 (fr) * 2018-12-10 2020-07-23 Denali Therapeutics Inc. Biomarqueurs de maladie lysosomale et leurs méthodes d'utilisation
CN113265461A (zh) * 2021-07-02 2021-08-17 北京华诺奥美医学检验实验室有限公司 一种用于检测高频基因致病变异的引物组和探针组及试剂盒
US11957713B2 (en) 2016-10-14 2024-04-16 Children's Medical Center Corporation Compositions and methods for treating diseases and disorders of the central nervous system

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11957713B2 (en) 2016-10-14 2024-04-16 Children's Medical Center Corporation Compositions and methods for treating diseases and disorders of the central nervous system
WO2018136435A1 (fr) * 2017-01-17 2018-07-26 Children's Medical Center Corporation Compositions et procédés de traitement de maladies et de troubles du stockage lysosomial
WO2018136434A1 (fr) * 2017-01-17 2018-07-26 Children's Medical Center Corporation Compositions et méthodes de diagnostic et de traitement des maladies peroxysomales
CN110913872A (zh) * 2017-01-17 2020-03-24 儿童医疗中心有限公司 治疗溶酶体贮积疾病及病症的组合物和方法
US11548936B2 (en) 2017-01-17 2023-01-10 Children's Medical Center Corporation Compositions and methods for treating lysosomal storage diseases and disorders
CN110913872B (zh) * 2017-01-17 2023-08-04 儿童医疗中心有限公司 治疗溶酶体贮积疾病及病症的组合物和方法
AU2018210853B2 (en) * 2017-01-17 2023-09-28 Children's Medical Center Corporation Compositions and methods for treating lysosomal storage diseases and disorders
US12000843B2 (en) 2017-01-17 2024-06-04 Children's Medical Center Corporation Compositions and methods for diagnosing and treating peroxisomal diseases
WO2020123511A3 (fr) * 2018-12-10 2020-07-23 Denali Therapeutics Inc. Biomarqueurs de maladie lysosomale et leurs méthodes d'utilisation
CN113826015A (zh) * 2018-12-10 2021-12-21 戴纳立制药公司 溶酶体贮积症的生物标志物及其使用方法
CN113265461A (zh) * 2021-07-02 2021-08-17 北京华诺奥美医学检验实验室有限公司 一种用于检测高频基因致病变异的引物组和探针组及试剂盒

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