U.S. patent application number 14/775507 was filed with the patent office on 2016-01-28 for hyposialylation disorders.
This patent application is currently assigned to EMORY UNIVERSITY. The applicant listed for this patent is EMORY UNIVERSITY, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SER, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SER. Invention is credited to Nuria Carrillo-Carrasco, William A. Gahl, Miao He, Marjan Huizing, Rong Jiang, Xueli Li.
Application Number | 20160025717 14/775507 |
Document ID | / |
Family ID | 50680134 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160025717 |
Kind Code |
A1 |
Huizing; Marjan ; et
al. |
January 28, 2016 |
HYPOSIALYLATION DISORDERS
Abstract
Methods are disclosed for diagnosing a hyposialylation disorder.
Methods are also disclosed for determining the effectiveness of a
therapeutic agent for treatment of a hyposialylation disorder in a
subject. These methods include measuring an amount of
monosialylated Thomsen-Friedenreich (ST) antigen and measuring an
amount of non-sialylated Thomsen-Friedenreich antigen (T) in a
biological sample, such as a serum or plasma sample from the
subject, and determining the ratio of T to ST.
Inventors: |
Huizing; Marjan; (Santa
Cruz, CA) ; Gahl; William A.; (Kensington, MD)
; Carrillo-Carrasco; Nuria; (Bethesda, MD) ; He;
Miao; (Alpharetta, GA) ; Li; Xueli; (Tucker,
GA) ; Jiang; Rong; (Tucker, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY,
DEPARTMENT OF HEALTH AND HUMAN SER
EMORY UNIVERSITY |
Bethesda
Atlanta |
MD
GA |
US
US |
|
|
Assignee: |
EMORY UNIVERSITY
Atlanta
GA
THE UNITED STATES OF AMERICA, as represented by the Secretary,
Department of Health and Human Serv
Bethesda
MD
Emory University
Atlanta
GA
|
Family ID: |
50680134 |
Appl. No.: |
14/775507 |
Filed: |
March 13, 2014 |
PCT Filed: |
March 13, 2014 |
PCT NO: |
PCT/US2014/025633 |
371 Date: |
September 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61785094 |
Mar 14, 2013 |
|
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|
Current U.S.
Class: |
514/23 ; 435/29;
435/7.1; 435/7.92; 436/501; 436/94 |
Current CPC
Class: |
G01N 33/6893 20130101;
G01N 33/5308 20130101; G01N 2800/04 20130101; A61K 45/06
20130101 |
International
Class: |
G01N 33/53 20060101
G01N033/53; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method for diagnosing a hyposialylation disorder in a subject,
comprising measuring an amount of monosialylated
Thomsen-Friedenreich (ST) antigen and measuring an amount of
non-sialylated Thomsen-Friedenreich antigen (T) in a biological
sample from the subject, wherein Thomsen-Friedenreich antigen is
N-acetylgalactosamine (GalNAc) linked to galactose (Gal); and
determining the ratio of T to ST; wherein a ratio of T to ST of
about 0.052 or greater diagnoses the subject as having the
hyposialylation disorder.
2. The method of claim 1, further comprising administering to the
subject a therapeutic agent for the treatment of the
hyposialylation disorder if the ratio of T to ST is greater than
about 0.052.
3. The method of claim 1, wherein a ratio of T to ST of about 0.06
or greater diagnoses the subject as having the hyposialylation
disorder.
4. The method of claim 3, further comprising administering to the
subject a therapeutic agent for the treatment of the
hyposialylation disorder if the ratio of T to ST is greater than
about 0.06.
5. A method of determining the effectiveness of a first dosage of a
therapeutic agent for treatment of a hyposialylation disorder in a
subject, comprising measuring monosialylated Thomsen-Friedenreich
(ST) antigen and measuring non-sialylated Thomsen-Friedenreich
antigen (T) in a biological sample from the subject, wherein
Thomsen-Friedenrich antigen is N-acetylgalactosamine (GalNAc)
linked to galactose (Gal); and determining the ratio of T to ST;
wherein a ratio of T to ST of less than about 0.06 indicates that
the first dosage of the therapeutic agent is effective for the
treatment of the hyposialylation disorder, and a ratio of T to ST
of about 0.06 or greater indicates that the first dosage of the
therapeutic agent is not effective for the treatment of the
hyposialylation disorder.
6. The method of claim 5, further comprising administering to the
subject a second dosage of the therapeutic agent, wherein when a
ratio of T to ST of less than about 0.06 indicates that the second
dosage of the agent is effective for the treatment of the subject,
and wherein when a ratio of T to ST of about 0.06 or greater,
indicates that the second dosage of the therapeutic agent is not
effective for the treatment of the subject.
7. A method of determining the effectiveness of a first dosage of a
therapeutic agent for treatment of a hyposialylation disorder in a
subject, comprising measuring monosialylated Thomsen-Friedenreich
(ST) antigen and measuring non-sialylated Thomsen-Friedenreich
antigen (T) in a biological sample from the subject, wherein
Thomsen-Friedenrich antigen is N-acetylgalactosamine (GalNAc)
linked to galactose (Gal); and determining the ratio of T to ST;
wherein a ratio of T to ST of less than about 0.052 indicates that
the first dosage of the therapeutic agent is effective for the
treatment of the hyposialylation disorder, and a ratio of T to ST
of about 0.052 or greater indicates that the first dosage of the
therapeutic agent is not effective for the treatment of the
hyposialylation disorder.
8. The method of claim 3, further comprising administering to the
subject a second dosage of the therapeutic agent, wherein a ratio
of T to ST of less than about 0.052 indicates that the second
dosage of the agent is effective for the treatment of the subject,
and wherein a ratio of T to ST of about 0.052 or greater indicates
that the second dosage of the therapeutic agent is not effective
for the treatment of the subject.
9. The method of claim 2, wherein the therapeutic agent is
N-acetyl-D-mannosamine (ManNAc), N-acetylneuraminic acid (Neu5Ac),
sialic acid, mannosamine, or one or more sialylated compounds.
10. The method of claim 9, wherein the one or more sialylated
compounds comprises intravenous immunoglobulin (IVIG) or
sialyllactose.
11. The method of claim 5, wherein the first dosage of the
therapeutic agent and the second dosage of the therapeutic agent
are different.
12. The method of claim 2, wherein the therapeutic agent is an
extended release formulation or is encapsulated.
13. The method of claim 1, wherein the hyposialylation disorder is
GNE myopathy.
14. The method of claim 1, wherein the hyposialylation disorder is
a congenital disorder of glycosylation.
15. The method of claim 1, wherein the hyposialylation disorder
comprises renal hyposialylation.
16. The method of claim 1, wherein the hyposialylation disorder
comprises podocytopathy, glomerular disease, focal segmental
glomerulosclerosis, or diabetic nephropathy.
17. The method of claim 1, wherein the hyposialylation disorder is
a muscular dystrophy, liver disorder, kidney disorder, or sleep
disorder.
18. The method of claim 1, wherein the hyposialylation disorder is
a neurodegenerative disorder, which comprises disorders of
.beta.-amyloid accumulation in the brain.
19. The method of claim 1, wherein detecting monosialylated
Thomsen-Friedenreich (ST) antigen and non-sialylated
Thomsen-Friedenreich (T) antigen comprises using mass spectrometry
to detect the mass transition between a parent ion and fragment ion
of the T antigen and a parent ion and fragment ion of the ST
antigen.
20. The method of claim 19, wherein (a) a parent ion of the T
antigen is 534, a parent ion of the monosialylated ST antigen is
895, and the fragment ions are 298 and 520, respectively; and/or b)
wherein multiple reaction monitoring (MRM) transitions for
T-antigen is m/z 534/298 and for monosialylated ST-antigen is m/z
895/520.
21. The method of claim 19, wherein detecting the mass transition
using mass spectrometry provides the relative ratio of T to
monosialylated ST antigens.
22. The method of claim 19, wherein the mass spectrometry is
MALDI-TOF mass spectrometry and/or LC-mass spectrometry.
23. The method of claim 22, wherein the method comprises releasing
O-glycans from the biological sample; desalting the O-glycans; and
permethylating the O-glycans prior to MALDI-TOF mass
spectrometry.
24. The method of claim 23, wherein releasing O-glycans comprises
treating the biological sample with sodium hydroxide and sodium
borohydrate.
25. The method of claim 23, wherein desalting the O-glycans
comprises using ion-exchange chromatography.
26. The method of claim 1, wherein the biological sample is a
plasma sample or a serum sample.
27. The method of claim 1, wherein the biological sample is a
biopsy sample or a cell sample.
28. The method of claim 1, further comprising purifying
glycoproteins from the biological sample.
29. The method of claim 1, wherein the control is the ratio of T to
ST in a sample from a subject known not to have a sialylation
disorder.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This claims the benefit of U.S. Provisional Application No.
61/785,094, filed Mar. 14, 2013, which is incorporated herein by
reference.
FIELD OF THE DISCLOSURE
[0002] This relates to the field of hyposialylation disorders,
specifically to methods for diagnosing these disorders using the
ratio of Thomsen-Friedenreich antigen (T) to monosialylated
Thomsen-Friedenreich antigen (ST).
BACKGROUND
[0003] Sialic acid contains a net negative charge and is found on
terminating branches of glycans, which include glycoproteins (with
N- or O-linked glycosylation) and glycolipids (including
glycosphingolipids or gangliosides). The sialic acid modification
of cell surface molecules impacts protein structure and stability,
regulation of cell adhesion, and signal transduction, amongst other
processes.
[0004] Clinical diseases with a reduced amount of sialic acid bound
to glycans are called "hyposialylation disorders." Hyposialylation
can occur in a specific tissue or can be systemic. In some cases
genetic defects cause hyposialylation disorders, but the etiology
of many of these disorders is unknown.
[0005] One hyposialylation disorder associated with a genetic
defect is GNE myopathy (also called HIBM, IBM type 2, Nonaka
myopathy, or Distal Myopathy with Rimmed Vacuoles (DMRV)). GNE
myopathy is caused by mutations in the GNE gene, encoding the key
enzyme in sialic acid synthesis, the bifunctional enzyme
UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase.
Decreased GNE enzyme activity is believed to reduce sialic acid
levels.
[0006] Other genetic disorders that may involve sialylation defects
are the congenital disorders of glycosylation (CDGs). CDGs are a
group of human genetic disorders characterized by alterations in
glycoconjugates (Jaeken, J Inherit Metab Dis 2011, 34:853-858). A
majority of the CDGs are caused by primary defects in the N- and/or
O-glycosylation pathways that lead to defective glycan
biosynthesis. In the past decade, about 60 genetic diseases have
been identified that alter glycan synthesis and structure and
ultimately the functions of many organ systems (He et al., The
congenital disorders of glycosylation. In: Laboratory Diagnosis of
inherited Metabolic Diseases. edn. Edited by Garg et al.,
Washington, D.C.: AACC Press; 2012: 179-199). CDG type I (CDG-I)
disorders result from impaired synthesis of glycans, which may lead
to unoccupied glycosylation sites on glycoproteins and glycolipids.
CDG type II (CDG-II) disorders result from impaired processing of
glycans, which lead to accumulation of glycoproteins and
glycolipids with abnormal structures. CDG-II disorders also include
defects in chaperones and Golgi-trafficking complexes, such as
defects in the conserved oligomeric Golgi complex (COG), dolichol
synthesis, and CMP-sialic acid synthesis, which impair multiple
glycosylation pathways including both N- and O-glycan synthesis and
N-glycan processing (He et al., supra). Some multiple glycosylation
defects may also present as mixed CDG-I and II (Perez et al., JIMD,
epub 2012; Perez et al., JIMD 2011, 1: 117-123; Mandato et al.,
Pediatr Res 2006, 59(2):293-298).
[0007] There is a need for non-invasive methods for the diagnosis
of glycosylation disorders, including hyposialylation disorders,
such as GNE myopathy and CDG disorders, and methods to determine
the effectiveness of therapeutic agents for the treatment of
hyposialylation disorders.
SUMMARY OF THE DISCLOSURE
[0008] Methods are disclosed for diagnosing a hyposialylation
disorder. These methods include measuring an amount of
monosialylated Thomsen-Friedenreich (ST) antigen, and measuring an
amount of non-sialylated Thomsen-Friedenreich antigen (T) in a
biological sample from the subject and determining the ratio of T
to monosialylated ST.
[0009] In some embodiments, a ratio of T to monosialylated ST of
about 0.06 or higher in a biological sample, such as a plasma or
serum sample diagnoses the hyposialylation disorder. In other
embodiments, a ratio of T to monosialylated ST of about 0.052 or
higher in a biological sample, such as a plasma or serum sample
diagnoses the hyposialylation disorder.
[0010] In other embodiments, the methods can include determining
whether a subject will respond to a specific therapeutic agent,
such as an agent that increases sialylation. In some examples, the
methods include administering to the subject a therapeutic agent
for the treatment of the hyposialylation disorder if the T to
monosialylated ST in a plasma or serum sample is 0.06 or higher. In
other examples, the methods include administering to the subject a
therapeutic agent for the treatment of the hyposialylation disorder
if the T to monosialylated ST in a plasma or serum sample is 0.052
or higher. The sample can be a plasma sample, a serum sample, a
tissue sample or a cell extract.
[0011] In additional embodiments, a ratio of T to ST of less than
about 0.06 indicates that the therapeutic agent is effective for
the treatment of the hyposialylation disorder. In other
embodiments, a ratio of T to ST of about 0.06 or greater indicates
that the first dosage of the therapeutic agent is not effective for
the treatment of the hyposialylation disorder. In further
embodiments, a ratio of T to ST of less than about 0.052 indicates
that the therapeutic agent is effective for the treatment of the
hyposialylation disorder. In other embodiments, a ratio of T to ST
of about 0.052 or greater indicates that the first dosage of the
therapeutic agent is not effective for the treatment of the
hyposialylation disorder.
[0012] In yet other embodiments, these methods can be used to
determine the lowest effective dosage of the therapeutic agent of
use to treat the subject. The sample can be a plasma sample, a
serum sample, a tissue sample or a cell extract. In additional
embodiments, these methods can be used to determine the lowest
effective dosage of the therapeutic agent of use to treat the
subject. The sample can be a plasma sample, a serum sample, a
tissue sample or a cell extract.
[0013] In some embodiments, methods are also disclosed for
determining the effectiveness of a therapeutic agent for treatment
of a hyposialylation disorder in a subject. These methods include
measuring monosialylated Thomsen-Friedenreich (ST) antigen and
measuring non-sialylated Thomsen-Friedenreich antigen (T) in a
sample from the subject and determining the ratio of T to ST.
[0014] In some embodiments, the method is non-invasive. In
specific, non-limiting examples of any of the methods disclosed
herein, the sample is a plasma or serum sample. In other
embodiments of any of the methods disclosed herein, this sample can
be another tissue or cell sample, including but not limited to
platelet, white cell, red cell, cerebrospinal fluid, urine, biopsy
material from liver, kidney or muscle.
[0015] The foregoing and other features and advantages of the
invention will become more apparent from the following detailed
description of a several embodiments which proceeds with reference
to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A-1B: Plasma O-glycan MALDI profile and quantitative
comparison between 10 normal controls and 3 patients with Conserved
Oligomeric Golgi (COG) deficiency. FIG. 1A: Human normal plasma
O-glycans were released by .beta.-elimination and permethylated
before the MALDI-TOF analysis. Measured m/z of each O-glycan are
shown as well as their structures. FIG. 1B: the comparison of
relative concentration of O-glycans between healthy control
subjects and COG patients (compared to an internal standard (I.S.),
raffinose at m/z 681). Control (n=10, shown in triangles), COG7
(n=2, shown in circles), COG4 (n=1, shown in circles).
Monosaccharides in drawings of glycan structures: Black
squares=GlcNAc, Grey circles=Gal, Black diamonds=Neu5Ac, Grey
squares=GalNAc.
[0017] FIGS. 2A-2D: Plasma O-glycan LC-MSMS quantification and
comparison study between 40 healthy control subject and 6 CDG-II
patient sera. FIG. 2A: Chromatograph of multiple reaction
monitoring (MRM) transitions for T-antigen (m/z 534/298) and
monosialylated ST-antigen (m/z 895/520). FIG. 2B: MRM triggered
enhanced product ion (EPI) profiles of T-antigen (top) and
monosialylated-ST-antigen (bottom). Multiple specific B and Y ion
fragments were identified from these two O-glycan species in plasma
total glycoprotein from normal controls, which confirms the
specificity of the MRM transitions for each glycan. FIG. 2C:
Linearity study of T-antigen quantification from a concentration
range of 0.0625-5 .mu.M. The correlation coefficient between the
known standard concentration and measured concentration of
T-antigen, R.sup.2 is 1, with slope at 0.87. FIG. 2D: Comparison of
T-antigen, monosialylated ST-antigen and T-antigen/monosialylated
ST-antigen ratio (T/ST) between 40 healthy control plasma samples
and 6 plasma samples from different CDG-II patients including two
patients with COG7, one with COG4 and two with mixed CDG-I and
CDG-II disease. Controls are shown in mixed color and shape, and
the six patients shown as triangles. Dashed lines represent cutoffs
to separate patients from the normal controls.
[0018] FIG. 3. T to monosialylated ST Ratio as a biomarker for
HIBM/GNE myopathy. The structure of T and monosialylated ST are
provided at the top. Exemplary MALDI-TOF analyses are shown in the
left panels. A table of the ratios for a subset of patients is
provided on the right of the figure.
[0019] FIG. 4. NCAM (H-300) immunoblotting of serum glycoproteins.
Serum samples (20 .mu.g) from neuraminidase treated control (NA),
control (C-1, C-2), and GNE myopathy patients (see Table 6 for
details) were immunoblotted with NCAM antibodies (H-300; sc-10735).
Compared to control, serum from GNE myopathy patients showed a
slight downshift of the 140 kDa NCAM isoform. A similar downshift
was present in neuraminidase treated control serum (NA). Dotted
line is to aid in discerning migration. See FIG. 7B for the full
gel images.
[0020] FIG. 5. Muscle lectin histochemistry. Paraffin-embedded
muscle sections from biceps (control and GNE-28) and gastrocnemius
(GNE-21) were stained with three lectins (grey `lines`; membrane
staining) informative for sialylation status and co-stained with
the nuclear dye DAPI (grey `dots`; nuclear staining). GNE myopathy
muscle specimens show selective hyposialylation compared to control
muscle, demonstrated by apparent normal staining of WGA (binding to
most sialic acid groups), but decreased staining of SNA
(predominantly binding terminal .alpha.(2,6)-linked sialic acid on
all glycans). In addition, staining of VVA (predominantly binding
terminal GalNAc, without sialic acid attached, O-linked to serine
or threonine residues of glycoproteins) was increased in GNE
myopathy muscle specimen compared to control, indicating
hyposialylation of O-linked glycans. Specificity of the WGA, SNA
and VVA lectins is demonstrated in FIG. 8.
[0021] FIGS. 6A-6B. Plasma O-glycan MALDI-TOF/TOF profiles and
quantitative comparison of T and ST antigens of control and GNE
myopathy patients. FIG. 6A. Human control and GNE myopathy plasma
O-glycan species were released by .beta.-elimination and
permethylated before MALDI-TOF/TOF analysis. Measured m/z and %
intensity compared to the internal standard (I.S.) raffinose of the
major detected small O-glycan species are shown as well as their
structures (squares, GalNAc; circles, Gal; diamonds, Sia; squares,
GlcNAc). FIG. 6B. Comparison of concentrations of T-antigen,
monosialylated T-antigen (ST) and their ratio T/ST in plasma
(evaluated by LC-MS\MS) from 50 healthy controls (circles) and
different GNE myopathy patients (squares). Plasma values of a GNE
myopathy patient before (solid triangle) and after (open triangle)
IVIG therapy are indicated. Dashed lines represent cutoffs to
establish the normal range [.about.2.times. standard deviation (SD)
of the mean (0.033)]. For additional information see Tables 5 and
6.
[0022] FIGS. 7A-7C. NCAM immunoblotting of serum glycoproteins.
Serum samples (20 .mu.g) from neuraminidase treated control (NA),
control (C-1, C-2), and GNE myopathy patients were immunoblotted
with the anti-NCAM antibodies (FIG. 7A) RNL-1 (sc-53007) and (FIG.
7B) H-300 (sc-10735) as described by Valles-Ayoub et al. Genet.
Test. Mol. Biomarkers 16(5), 313-317 (2012). FIG. 7A.
Immunoblotting with the NCAM RNL-1 antibody does not show an
apparent different banding pattern in serum of GNE myopathy
patients compared to control serum. Neuraminidase treated control
serum did not show a different banding pattern. FIG. 7B-7C.
Immunoblotting with the NCAM H-300 antibody (see also FIG. 4),
showed a slight downshift of the 140 kDa NCAM isoform in serum of
GNE myopathy patients compared to control serum. A similar
downshift was present in neuraminidase treated control serum (NA).
Dotted line is to aid in discerning migration.
[0023] FIG. 8A-8B. Control histochemistry for WGA, SNA and VVA
lectin specificity. Due to limited availability of control human
muscle slides, wild type mouse (C57BL/6 strain) muscle
(gastrocnemius and gluteus) slides were used to test specificity of
the WGA, SNA lectins (used in FIG. 2). For specificity of VVA, GNE
myopathy patient (GNE-21) muscle slides were used (since VVA does
not bind to wild type mouse muscle glycans). FIG. 8A.
Representative images of paraffin embedded muscle slides stained
with each lectin as well as with substrate-inhibited lectin (grey
`lines`; membrane staining) and with the nuclear dye DAPI (grey
`dots`; nuclear staining). Each FITC-labeled lectin was incubated
with its specific inhibitory carbohydrate (i.e., Neu5Ac for WGA and
SNA, GalNAc for VVA) prior to incubation on muscle slides. Note
that sugar-inhibited lectins (right panels) show a greatly reduced
or absent fluorescent signal for each lectin compared to the
original lectin signal (left panels). FIG. 8B. Representative
images of paraffin embedded control muscle slides either untreated
(-NA) or treated/desialylated with neuraminidase (+NA). Both the
SNA and WGA signals greatly decreased after neuraminidase
incubation, indicating de-sialylation of the tissue glycans and
specificity of the lectins. A neuraminidase-inhibition control was
not provided for VVA, since sialylated O-GalNAc (STn-antigen) is
not present in normal muscle tissue (only in disease tissue).
[0024] FIG. 9A-9D. Western blotting followed by lectin staining of
serum glycoproteins. Control (C-1, C-2), Neuraminidase treated
control (NA), and GNE myopathy patients (GNE-2, -5, -10, -13, -16)
serum was electrophoresed on SDS-PAGE gels, followed by
electroblotting on nitrocellulose membranes. The membranes were
incubated with the lectins WGA (FIG. 9A), SNA (FIG. 9B) or VVA
(FIG. 9C). 10 .mu.g of total serum protein was loaded in the WGA
and SNA labeled blots, and 20 .mu.g total serum protein was loaded
in the VVA labeled blot. The NA-treated control samples showed the
expected reduction (for WGA and SNA) or increase (for VVA) in
lectin binding, no significant differences in binding were present
in GNE myopathy patients' compared to control samples. The Ponceau
S (FIG. 9D) stained membrane is an image from the blot before SNA
labeling, and serves as a representative image for protein loading
control of all blots (each blot was loaded with the same samples).
The positive Ponceau S signal in the NA-treated lane and the
absence of SNA staining in this lane, indicating total
desialylation of NA-treated serum as well as specificity of the SNA
lectin.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
[0025] Methods are disclosed for diagnosing a hyposialylation
disorder, including, but not limited to, a congenital disorder of
glycosylation and GNE myopathy. Methods are also disclosed for
determining the effectiveness of a therapeutic agent for treatment
of a hyposialylation disorder in a subject. These methods include
measuring an amount of monosialylated Thomsen-Friedenreich (ST)
antigen, and measuring an amount of non-sialylated
Thomsen-Friedenreich antigen (T) in a biological sample from the
subject and determining the ratio of T to ST.
[0026] In additional embodiments, these methods can be used to
determine the lowest effective dosage, or duration, of the
therapeutic agent of use to treat the subject. The methods can be
used to monitor the efficacy of a therapeutic agent.
TERMS
[0027] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8). In order to facilitate review of the various
embodiments of this disclosure, the following explanations of
specific terms are provided:
[0028] Alteration: A statistically significant change in a
parameter as compared to a control. In one example, an "increase"
is a statistically significant elevation in a parameter, such as
the presence of a biological marker, or the ratio of two biological
markers, such at the T/ST ratio. The alternation can be measured as
compared to a control. Suitable statistical analyses are well known
in the art, and include, but are not limited to, Student's T test
and ANOVA assays. In one example, a "decrease" or "reduction" is a
statistically significant decline in a parameter, such as the
presence of a biological marker, such as the T/ST ratio as compared
to a control. In another example, an "increase" is a statistically
significant higher level of a parameter, such as the presence of a
biological marker, such as the T/ST ratio as compared to a control.
Suitable statistical analyses are well known in the art, and
include, but are not limited to, Student's T test and ANOVA
assays.
[0029] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals.
[0030] Congenital disorders of glycosylation (CDG): A group of
disorders of abnormal glycosylation of glycans caused by deficiency
one of the different steps in the synthetic, transport and
metabolism pathways of glycans. Most commonly, the disorders begin
in infancy; manifestations range from severe developmental delay
and hypotonia with multiple organ system involvement to
hypoglycemia and protein-losing enteropathy with normal
development. However, most types have been described in only a few
individuals, and thus understanding of the phenotypes is limited.
In PMM2-CDG (CDG-Ia), the most common type reported, the clinical
presentation and course are highly variable, ranging from death in
infancy to mild involvement in adults.
[0031] CDG type I (CDG-I) disorders result from impaired synthesis
of glycans, which may lead to unoccupied glycosylation sites on
glycoproteins and glycolipids. CDG type II (CDG-II) includes
defects in processing of glycans, which lead to accumulation of
glycoproteins and glycolipids with abnormal structures. CDG-II
includes defects in chaperones and Golgi-trafficking complexes,
such as defects in the conserved oligomeric Golgi complex (COG),
dolichol synthesis, and CMP-sialic acid synthesis, which impair
multiple glycosylation pathways including both N- and O-glycan
synthesis and N-glycan processing. Some multiple glycosylation
defects may also present as mixed CDG-I and II. In addition, there
is a growing number of patients with strong evidence of a
glycosylation defect, whose molecular basis has not yet been
identified (CDG-IIx).
[0032] Control: A value used as a source for comparison with an
experimentally determined value. A control can be a standard value,
a ratio (such as of a T/ST ratio) from one subject, or averaged
from many subjects, who does not have a known disorder (such as a
hyposialylation disorder), or a baseline concentration obtained
from a subject at an earlier time point, prior to an onset of
symptoms.
[0033] Diabetic Nephropathy: A progressive kidney disease caused by
angiopathy of capillaries in the kidney glomeruli. Diabetic
nephropathy is characterized by nephrotic syndrome and diffuse
glomerulosclerosis due to longstanding diabetes mellitus, and is a
prime indication for dialysis. It is classified as a microvascular
complication of diabetes.
[0034] These subjects generally have macroalbuminuria (urinary
albumin excretion of more than 300 mg in a 24-hour collection) or
macroalbuminuria and abnormal renal function as represented by an
abnormality in serum creatinine, calculated creatinine clearance,
or glomerular filtration rate (GFR). Clinically, diabetic
nephropathy is characterized by a progressive increase in
proteinuria and decline in GFR, hypertension. Subjects with
diabetic nephropathy have a high risk of cardiovascular morbidity
and mortality.
[0035] Determining or Measuring: Identifying the presence of a
target molecule in a sample. There terms refer to measuring a
quantity or quantitating a target molecule in the sample, either
absolutely or relatively. For example, T and ST can be analyzed in
a sample from a subject of interest, such as a subject suspected of
having a hyposialylation disorder. The sample can be any biological
sample of interest, such as, but not limited to, a plasma sample,
serum sample, or tissue extract. Generally, detecting, measuring or
determining a biological molecule requires performing an assay,
such as mass spectrometry, and not simple observation.
[0036] Diagnosing or diagnosis of a hyposialylation disorder:
Detecting the disorder by measuring specific parameters. For
example, a hyposialylation disorder can be detected by determining
the T/ST ratio in a biological sample. Diagnosis can encompass
laboratory confirmation of a pre-existing clinical condition or a
specific disease.
[0037] Focal segmental glomerulosclerosis (FSGS): A nephrotic
syndrome of children, adolescents and adults that usually diagnosed
based on pathological findings in a kidney biopsy. Scarring of the
glomerulus is apparent, but some of the glomeruli are involved, and
only a part of each glomerulus is involved. Generally, tissue
sections show heavy PAS staining and IgM and C3 are present in the
sclerotic segments. FSGS presents as a nephrotic syndrome, which is
characterized by edema (associated with weight gain),
hypoalbuminemia, hyperlipidemia and hypertension. In adults it can
also present as kidney failure and proteinuria. The cause can be
genetic mutations in the FSGS1 (ACTN4, OMIM No. 603278), FSGS2
(TRPC6, OMIM No. 607832), FSGS3 (CD2AP, OMIM No. 607832), FSGS4
(APOL1, OMIM No. 612551), FSGS5 (IMF2, OMIM No. 613237) or SRN1
(NPHS2, OMMIM 600995) genes.
[0038] Glycoprotein: Proteins that contain oligosaccharide chains
(glycans) covalently attached to polypeptide side-chains. The
carbohydrate is attached to the protein in a cotranslational or
posttranslational modification process known as glycosylation.
There are two main types of glycosylation, N-glycosylation,
O-glycosylation. In N-glycosylation, the addition of the sugar
occurs on an amide nitrogen, such as in the side chain of
asparagine. In O-glycosylation, the addition of the sugar occurs on
a hydroxyl oxygen, such as on the side chain of hydroxylysine,
hydroxyproline, serine or threonine. The sugars commonly found in
eukaryotic glycoproteins include, but are not limited to,
.beta.-D-glucose, .beta.-D-glactose, .beta.-D-mannose,
.alpha.-L-fucose, N-Acetylgalactosamine, N-Acetylglucosamine,
N-Acetylneuraminic acid, and xylose.
[0039] Hereditary Inclusion Body Myopathy: A rare autosomal
recessive neuromuscular disorder, also called GNE myopathy (and
DMRV, Nonaka myopathy, IBM2 QIBM). (Argov, et al., Neurology 60,
1519-1523 (2003); Eisenberg, et al. (2001) Nat Genet 29, 83-87
(2001); Griggs, et al. (1995) Ann Neurol 38, 705-713 (1995)) that
is a hyosialylation disorder. The disease usually manifests at
approximately 20 years of age with foot drop and slowly progressive
muscle weakness and atrophy. The cranial nerves, sensation, and
mental acuity are all normal, and creatine kinase levels are
normal. Histologically, it is associated with muscle fiber
degeneration and formation of vacuoles containing 15-18 nm
tubulofilaments that immunoreact like .beta.-amyloid, ubiquitin,
prion protein and other amyloid-related proteins (see Askanas et
al. Curr Opin Rheumatol 10, 530-542 (1998); Nishino, et al. (2005)
Acta Myol 24, 80-83 (2005); Askanas, et al. Ann Neurol 34, 551-560
(1993); Argov, et al. Curr Opin Rheumatol 10, 543-547 (1998)). Both
weakness and histological changes initially spare the quadriceps.
However, the disease is relentlessly progressive, with patients
becoming incapacitated and wheelchair-confined within two to three
decades. GNE myopathy is caused by mutations in the GNE gene,
encoding the bifunctional key enzyme in sialic acid synthesis,
UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase.
Human GNE mutations result in lower activities of both epimerase
and kinase function of this enzyme. The decrease or absence of
these GNE enzymatic activities results in decreased de novo
production of sialic acid, causing hyposialylation of glycoprotein
and glycolipids, specifically a decrease in sialylation of O-linked
glycans. In GNE myopathy, hyposialylation is found mainly on
O-linked glycans in muscle.
[0040] Hyposialylation: Reduced or absent addition of sialic acid
(N-acetyl neuraminic acid (Neu5Ac) and its derivatives) to
galactose (Gal) or other underlying monosaccharides (such as, but
not limited to N-acetylgalactose (GalNAc)), Mannose (Man),
N-acetylglucosamine (GlcNAc), N-acetlylneuraminic acid (Neu5Ac) or
of sialic acid chains in polysialylation (PSA), such as on
PSA-NCAM.
[0041] Hyposialylation disorders are conditions with
hyposialylation of glycoproteins and glycolipids in affected
tissues. Hyposialylation of affected tissues can be detected, for
example, using histochemistry staining of fixed tissue slides with
specific lectins. A demonstration of a significant reduction (or
absence) of sialic acid, either by a statistically reduced
staining/binding of sialic acid recognizing lectins (such as, but
not limited to wheat germ agglutinin (WGA), Sambucus nigra
agglutinin (SNA), and Limax flavus agglutinin (LFA) or by presence
of staining of free monosaccharides underlying sialic acid on the
glycan chain, including galactose or GalNAc, by the lectins (such
as, but not limited to, helix pomatia agglutinin (HPA), Vicia
villosa agglutinin (VVA), jackfruit agglutinin (Jacalin), and
peanut agglutinin (PNA) can be used to identify hyposialylation
disorders, such as certain cases with myopathy (including the
adult-onset, progressive, autosomal recessive muscular disorder,
GNE myopathy, also called distal myopathy with rimmed vacuoles
(DMRV)/hereditary inclusion body myopathy (HIBM)), renal disorders
(including, but not limited to minimal change nephrosis, lupus
nephritis, IgA nephropathy), sleep disorders (including those with
reduced REM sleep), neurodegenerative disorders (including those
with amyloid depositions), cancers and liver disorders. Western
blotting or 2D gel electrophoresis followed by lectin labeling or
immunolabeling with a specific antibody to a sialoglycan can also
be used to detect hyposialylation disorders. Methods for detecting
are disclosed, for example, in Kakani et al. Am J Pathol 2012: 180:
1431-1440 and Niethamer et al. Mol Genet Metab 2012:
107:748-755).
[0042] Intravenous Immunoglobulin (IVIG): A blood product that
includes pooled polyvalent IgG extract from the plasma of a number
of blood donors. It is used as treatment for immune deficiencies
such as X-linked agammaglobulinemia, autoimmune diseases, such as
immune thrombocytopenia and Kawaski disease, and acute
infections.
[0043] Ion Exchange Chromatography: A chromatographic process that
allows the separation of ions and polar molecules based on their
charge. Ion-exchange chromatography retains analyte molecules on
the column based on coulombic (ionic) interactions. The stationary
phase surface displays ionic functional groups (R-X) that interact
with analyte ions of opposite charge. This type of chromatography
is further subdivided into cation exchange chromatography and anion
exchange chromatography. The ionic compound consisting of the
cationic species M+ and the anionic species B- can be retained by
the stationary phase.
[0044] Generally, a sample is introduced, either manually or with
an autosampler, into a sample loop of known volume. A buffered
aqueous solution (often called the "mobile phase") carries the
sample from the loop onto a column that contains a stationary phase
material that is typically a resin or gel matrix consisting of
agarose or cellulose beads with covalently bonded charged
functional groups. The target analytes (either anions or cations)
are retained on the stationary phase, but can be eluted by
increasing the concentration of a similarly charged species that
will displace the analyte ions from the stationary phase. For
example, in cation exchange chromatography, the positively charged
analyte can be displaced by the addition of positively charged
sodium ions. The analytes of interest are detected, such as by
conductivity or an ultraviolet (UV)/Visible light absorbance.
Generally, a chromatography data system (CDS) is used to control
the chromotography system.
[0045] Mass Spectrometry: A process used to separate and identify
molecules based on their mass. Mass spectrometry ionizes chemical
compounds to generate charged molecules or molecule fragments and
measures their mass-to-charge ratios. In a typical MS procedure, as
sample is ionized. The ions are separated according to their
mass-to-charge ratio, and the ions are dynamically detected by some
mechanism capable of detecting energetic charged particles. The
signal is processed into the spectra of the masses of the particles
of that sample. The elements or molecules are identified by
correlating known masses by the identified masses. "Time-of-flight
mass spectrometry" (TOFMS) is a method of mass spectrometry in
which an ion's mass-to-charge ratio is determined via a time
measurement. Ions are accelerated by an electric field of known
strength. This acceleration results in an ion having the same
kinetic energy as any other ion that has the same charge. The
velocity of the ion depends on the mass-to-charge ratio. The time
that it subsequently takes for the particle to reach a detector at
a known distance is measured. This time will depend on the
mass-to-charge ratio of the particle (heavier particles reach lower
speeds). From this time and the known experimental parameters one
can find the mass-to-charge ratio of the ion. "Liquid
chromatography-mass spectrometry" or "LC-MS" is a chemistry
technique that combines the physical separation capabilities of
liquid chromatography (or HPLC) with the mass analysis capabilities
of mass spectrometry. Liquid chromatography mass spectrometry
(LC-MS) separates compounds chromatographically before they are
introduced to the ion source and mass spectrometer. It differs from
gas chromatography (GC-MS) in that the mobile phase is liquid,
usually a mixture of water and organic solvents, instead of gas and
the ions fragments. Most commonly, an electrospray ionization
source is used in LC-MS.
[0046] Mean and Standard Deviation: The arithmetic mean is the
"standard" average, often simply called the "mean".
x _ = 1 n i = 1 n x i ##EQU00001##
The mean is the arithmetic average of a set of values.
[0047] The standard deviation (represented by the symbol sigma, a)
shows how much variation or "dispersion" exists from the mean. The
standard deviation of a random variable, statistical population,
data set, or probability distribution is the square root of its
variance. The standard deviation is commonly used to measure
confidence in statistical conclusions. Generally, twice the
standard deviation is about the radius of a 95 percent confidence
interval. Effects that fall far outside the range of standard
deviation are generally considered statistically significant. One
of skill in the art can readily calculate the mean and the standard
deviation from a population of values.
[0048] N-acetyl-D-mannosamine: The structure of
N-acetyl-mannosamine is.
##STR00001##
N-acetylmannosamine and derivatives thereof can also be used. The
structures of such N-acetylmannosamine derivatives useful in the
invention are defined by Formula I.
##STR00002##
wherein:
[0049] R.sub.1, R.sub.3, R.sub.4, or R.sub.5 is hydrogen, lower
alkanoyl, carboxylate or lower alkyl; and
[0050] R.sub.2 is lower alkyl, lower alkanoylalkyl, lower alkyl
alkanoyloxy. Derivates of N-acetylmanosamine are known; several
exemplary derivatives are disclosed below.
[0051] Neurodegenerative disorder: A disease or condition
associated with progressive loss of the structure or function of
neurons. Neurodegenerative disorders include, but are not limited
to Parkinson's disease, Alzheimer's disease and Huntington's
disease.
[0052] Alzheimer's disease is the most common form of dementia. In
the early stages, the most common symptom is difficulty in
remembering recent events. When Alzheimer's disease is suspected,
the diagnosis is usually confirmed with tests that evaluate
behavior and thinking abilities, often followed by a brain scan. As
the disease advances, symptoms can include confusion, irritability
and aggression, mood swings, trouble with language, and long-term
memory loss. Alzheimer's disease is characterized by loss of
neurons and synapses in the cerebral cortex and certain subcortical
regions. This loss results in gross atrophy of the affected
regions, including degeneration in the temporal lobe and parietal
lobe, and parts of the frontal cortex and cingulate gyms. Both
amyloid plaques and neurofibrillary tangles are clearly visible by
microscopy in brains of those afflicted by Alzheimer's disease.
Plaques are dense, mostly insoluble deposits of .beta.-amyloid
peptide and cellular material outside and around neurons. Tangles
(neurofibrillary tangles) are aggregates of the
microtubule-associated protein tau which has become
hyperphosphorylated and accumulate inside the cells themselves.
Although many older individuals develop some plaques and tangles as
a consequence of ageing, the brains of people with Alzheimer's
disease have a greater number of them in specific brain regions
such as the temporal lobe
[0053] Neurodegenerative diseases, such as, but not limited to,
Alzheimer's disease, can be associated with accumulation of
.beta.-amyloid in the brain. .beta.-amyloid is a polypeptide of
36-43 amino acids that is processed from the amyloid precursor
protein that is the main component of deposits found in the brains
of patients with Alzheimer's disease. Similar plaques appear in
some variants of Lewy body dementia and in inclusion body myositis
(a muscle disease), while .beta.-amyloid can also form the
aggregates that coat cerebral blood vessels in cerebral amyloid
angiopathy. The plaques are composed of a tangle of regularly
ordered fibrillar aggregates called amyloid fibers, a protein fold
shared by other peptides such as the prions associated with protein
misfolding diseases.
[0054] Amyloid also accumulates in muscle of patients with GNE
myopathy. Without being bound by theory, hyposialylation of amyloid
precursor protein (APP) has been proposed to be important in
misfolding and accumulation of amyloid, a process that may occur in
GNE myopathy muscle tissue and could be important in some
neurodegenerative diseases with brain amyloid accumulation.
[0055] Purified: The term "purified" does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified glycoprotein preparation is one in which the
glycoprotein referred to is more pure than the protein in its
natural environment within a cell. For example, a preparation of a
glycoprotein is purified such that the glycoprotein represents at
least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the total
biomolecule content of the preparation.
[0056] Renal hyposialylation disorder: A disease of the kidneys
characterized by decreased sialylation. In some subjects, the
glomeruli are hyposialylated. These disorders include some forms of
podocytopathies, minimal change nephrosis, focal and segmental
glomerulosclerosis, membranous glomerulonephritis, and other forms
of unexplained idiopathic nephrotic syndrome, as well as glomerular
basement membrane diseases such as Alport disease and thin membrane
disease. Such kidney disorders and conditions are sometimes
characterized by segmental splitting of the glomerular basement
membrane and/or podocytopathy due to disturbed polyanions on
podocyte membranes, or to changes in the amount or charge
(sialylation) of glomerular basement membrane components.
[0057] Sample: A biological specimen containing genomic DNA, RNA
(including mRNA), protein, glycoprotein, or combinations thereof,
obtained from a subject. In some examples, a sample is a bodily
fluid, such as, but not limited to, a blood, serum, or plasma
sample. A sample can be a cell or a tissue extract, such as, but
not limited to, platelets, red blood cells, or liver, muscle or
kidney biopsy (or cell) extracts. A bodily fluid is a natural
liquid or secretion of a subject's body, including cerebrospinal
fluid or urine.
[0058] Sialic acid: A negative charged sugar that is a terminal
sugar on glycans. The most common sialic acid is
5-N-acetylneuraminic acid, a monosaccharide with a nine-carbon
backbone. Other less common sialic acids are N- or O-substituted
derivatives of 5-N-neuraminic acid. Sialic acids are found widely
distributed in animal tissues and to a lesser extent in other
species, ranging from plants and fungi to yeasts and bacteria,
mostly in glycoproteins and gangliosides. The amino group generally
bears either an acetyl or glycolyl group. The hydroxyl substituents
include acetyl, lactyl, methyl, sulfate, and phosphate groups.
Sialic acid is transferred to an oligosaccharide by a
sialyltransferase.
[0059] In renal functions, sialic acid residues are important for
maintenance of glomerular integrity, facilitating glomerular
filtration, and their deficiency is implicated in proteinuria
and/or hematuria. It has also been reported that glomerular
podocyte and podocyte foot process morphologies are maintained by
the anionic charge of sialic acid residues on podocyte
glycoproteins and glycolipids, and that a barrier to protein
permeability is controlled by functional endothelial glycocalyx,
rich in sialic acid.
[0060] Sleep disorder: A medical disorder of the sleep patterns of
a person or animal. Some sleep disorders are serious enough to
interfere with normal physical, mental and emotional functioning.
Polysomnography is a test commonly used to diagnose some sleep
disorders. Sleep disorders include primary insomnia, bruxism,
delayed sleep phase syndrome, hyopnea syndrome, nacrcolepsy,
catalplexy, night tenors, parasomnia, periodic limb movement
disorder (PLMD), rapid eye movement (REM) behavior disorders,
restless leg syndrome, sleep apnea, sleep paralysis, sleepwalking,
nocturia, or somniphobia. Sleep disorders associated with
hyposialylation include sleep volume reductions and sleep quality
reductions, the former manifesting themselves as increased sleep
onset time, inadequate sleep time due to premature arousal and the
like, and the latter developing as symptoms such as bedtime shifts,
decreased deep sleep (non-REM sleep), sleep interruptions due to
premature arousal, and naps in active time zones. Sleep disorders
occur irrespective of the patient's age; especially the quality of
sleep decreases with aging (see U.S. Published Patent Application
No. 2011/0212917, incorporated herein by reference).
[0061] Diagnosis can be made by a test consisting of a plurality of
inquiries, and is established by electroencephalography or by
polysomnography, which measures multiple parameters, including
electroencephalograms. Diagnoses can be classified according to
internationally recognized criteria (The International
Classification of Sleep Disorder, ICSD).
[0062] Rapid eye movement sleep behavior disorder (RBD) is a sleep
disorder that involves abnormal behavior during the sleep phase
with rapid eye movement (REM sleep). The major and abnormal feature
of RBD is loss of muscle atonia (paralysis) during otherwise intact
REM sleep. This is the stage of sleep in which most vivid dreaming
occurs. The loss of motor inhibition leads to a wide spectrum of
behavioral release during sleep. This extends from simple limb
twitches to more complex integrated movement, in which sufferers
appear to be unconsciously acting out their dreams. These behaviors
can be violent in nature and in some cases will result in injury to
either the patient or their bed partner. Sleep disorders are
disclosed in U.S. Published Patent Application No. 2011/0212917,
which is incorporated herein by reference.
[0063] Standard: A substance or solution of a substance of known
amount, purity or concentration that is useful as a control. A
standard can also be a known value or concentration of a particular
substance. A standard can be compared (such as by spectrometric,
chromatographic, spectrophotometric, or statistical analysis) to an
unknown sample (of the same or similar substance) to determine the
presence of the substance in the sample and/or determine the
amount, purity or concentration of the unknown sample. In one
embodiment, a standard is a particular T/ST ratio. In another
embodiment, a standard is a known ratio of T/ST that is found in a
sample from a subject that does not have a hyposialylation
disorder.
[0064] Subject: Living organisms susceptible to hyosialylation
disorders; a category that includes both human and non-human
mammals, such as non-human primates.
[0065] Thomsen-Friedenreich Antigen: N-actetyl galactosamine linked
Galactose (Gal.beta.1-3GalNAc.alpha.1), also known as "T" antigen.
The monosialylated form of this antigen (Neu5Ac-Gal-GalNAc) is
called "ST" antigen; a disialylated form also exists. The
structures of T and ST are shown in FIG. 3.
[0066] Therapeutic agent: A molecule, such as a chemical compound,
antibody, small molecule, nucleic acid, protein, oligosaccharide,
or glycoprotein used for the treatment of a disorder.
[0067] Under conditions sufficient for: A phrase that is used to
describe any environment that permits the desired activity.
[0068] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. The term "comprises"
means "includes." All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including explanations of terms, will control. In addition, the
materials, methods, and examples are illustrative only and not
intended to be limiting.
Methods
[0069] Methods are disclosed herein for diagnosing a
hyposialylation disorder, including confirming a diagnosis of a
hyposialylation disorder, such as a clinical diagnosis. These
methods include obtaining a biological sample from a subject of
interest, such as a subject suspected of having a hyposialylation
disorder, and measuring monosialylated Thomsen-Friedenreich (ST)
antigen and measuring non-sialylated Thomsen-Friedenreich antigen
(T). The ratio of T to ST (T/ST), in the biological sample is
determined.
[0070] In some embodiments, a method is provided for diagnosing a
hyposialylation disorder, for example confirming the diagnosis of a
hyposialylation disorder, that includes measuring an amount of
monosialylated Thomsen-Friedenreich (ST) antigen in a biological
sample from the subject and measuring an amount of non-sialylated
Thomsen-Friedenreich antigen (T) in the biological sample from the
subject. The ratio of T to ST is determined. The ratio of T to ST
can be compared to a control, such as a standard value. In some
embodiments, a subject is selected that is suspected of having the
disorder, such as based on clinical symptoms.
[0071] In some embodiments, a ratio of T to monosialylated ST
(T/ST) in a plasma or serum sample of greater than about 0.051 to
greater than about 0.062, greater than about 0.052 to greater than
about 0.06, greater than about 0.058 to greater than about 0.062,
such as about 0.052 or greater, about 0.053 or greater, about 0.054
or greater, about 0.055 or greater, about 0.056 or greater, about
0.057 or greater, about 0.058 or greater, about 0.059 or greater,
or about 0.06 or greater indicates that the overall sialylation of
O-linked glycoproteins in the plasma or serum is below 95% of the
population and that the subject has the hyposialylation disorder,
and/or confirms the diagnosis of the hyposialylation disorder. In
some embodiments, a T/ST ratio in serum or plasma of greater than
about 0.07, about 0.08, about 0.09, or about 0.1 indicates that the
subject has a hyposialylation disorder. In other embodiments, a
ratio of T to ST (T/ST) of less than about 0.06, less than about
0.059, less than about 0.058, less than about 0.057, less than
about 0.056, less than about 0.054, less than about 0.053, less
than about 0.052, or less than about 0.051, in plasma or serum
indicates that the subject does not have the hyposialylation
disorder. In some embodiments, a ratio of T to ST in plasma or
serum of less than about 0.05, about 0.04 or about 0.03 indicates
that the subject does not have a hyposialylation disorder. In this
context, "about" indicates within about 0.005.
[0072] In some specific non-limiting examples, the biological
sample is a serum or plasma sample. Exemplary results showing the
establishment of the about 0.06 cutoff value for patients with a
hyposialylation disorder, and validation with patients plasma
samples are provided in FIG. 2D and FIG. 6B. Exemplary results
showing the establishment of the about 0.052 cut off value for
patients without a hyposialylation disorder is provided in FIG. 6B,
and a cut off value of 0.058 is shown in FIG. 2D.
[0073] In some embodiments, the methods also include administering
to the subject a therapeutic agent for the treatment of the
hyposialylation disorder, such as if the ratio of T to ST in a
serum or plasma sample from the subject is greater than about 0.051
to greater than about 0.062, greater than about 0.052 to greater
than about 0.06, greater than about 0.058 to greater than about
0.062, such as about 0.052 or greater, about 0.053 or greater,
about 0.054 or greater, about 0.055 or greater, about 0.056 or
greater, about 0.057 or greater, about 0.058 or greater, about
0.059 or greater, or about 0.06 or greater. Suitable therapeutic
agents are disclosed below.
[0074] A ratio of T to monosialylated ST (T/ST) can also be
measured in biological samples other than serum or plasma,
including, but not limited to platelets, red cells, white cells,
cerebrospinal fluid, cell extracts (such as cell culture extracts)
urine or a biopsy sample, such as a liver biopsy, muscle biopsy or
kidney biopsy. In some embodiments, T and monosialylated ST are
measured in biological samples from subjects known not to have the
hyposialylation disorder, and a control ratio of the T to ST is
established. T and ST are measured in a biological sample from a
subject of interest, to determine if the subject has the
hyposialylation disorder. In some embodiments, a T to ST ratio of
greater than two standard deviations greater than the control ratio
of T to ST diagnoses the hyposialylation disorder. In additional
embodiments, a ratio of T to ST of greater than three standard
deviations than the control ratio of T to ST diagnoses the
hyposialylation disorder. In some embodiments, the methods also
include administering to the subject a therapeutic agent for the
treatment of the hyposialylation disorder, such as if the T to ST
in a tissue sample other than serum or plasma is greater than two
standard deviations, such as three standard deviations greater than
the ratio of T to ST for the control, such as the mean T/ST ratio
for biological samples from subjects without the hyposialylation
disorder (and/or without any sialylation disorder). Suitable
therapeutic agents are disclosed below.
[0075] Methods are also disclosed herein for determining the
effectiveness of a first dosage, or the duration of a dosage, of a
therapeutic agent for treatment of a hyposialylation disorder in a
subject. The method can determine if a therapeutic agent of
interest is of use for treating the hyposialylation disorder in a
subject, or if the therapeutic agent has been administered for a
sufficient period of time to treat the subject. The methods can be
used to determine the lowest effective therapeutic dosage of an
agent for the treatment of a subject. These methods include
measuring monosialylated ST antigen and T antigen in a biological
sample from the subject administered the therapeutic agent. In some
embodiments, the methods include administering the therapeutic
agent to the subject. The ratio of T to monosialylated ST is
determined.
[0076] In some embodiments, a ratio of T to ST in a plasma or serum
sample of less than about 0.06, less than about 0.059, less than
about 0.058, less than about 0.057, less than about 0.056, less
than about 0.054, less than about 0.053, less than about 0.052, or
less than about 0.051 indicates that the first dosage of the
therapeutic agent is effective for the treatment of the
hyposialylation disorder, and/or that the therapeutic agent has
been administered for a sufficient duration of time to treat the
subject. In additional embodiments, a ratio of T to ST of less than
about 0.05, about 0.04 or about 0.03 indicates that the first
dosage of the therapeutic agent is effective for the treatment of
the hyposialylation disorder, and/or that the therapeutic agent has
been administered for a sufficient duration of time to treat the
subject. Biological samples other than serum or plasma can also be
used.
[0077] In additional embodiments, a ratio of T to ST of greater
than about 0.051, greater than about 0.052, greater than about
0.053, greater than about 0.054, greater than about 0.055, greater
than about 0.056, greater than about 0.057 or greater, greater than
about 0.058, greater than about 0.059, or greater than about 0.06,
such as in serum or plasma, indicates that the first dosage of the
therapeutic agent is not effective for the treatment of the
hyposialylation disorder and/or that the therapeutic agent has not
been administered for a sufficient duration of time to treat the
subject. In some embodiments, a serum or plasma T/ST ratio of
greater than about 0.07, about 0.08, about 0.09, or about 0.1
indicates that the first dosage of the therapeutic agent is not
effective for treating the subject, and/or that the therapeutic
agent has not been administered for a sufficient duration of time
to treat the subject. Biological samples other than serum or plasma
can also be used. In some non-limiting examples, for any of the
methods disclosed herein, the biological sample can be a sample
other than serum or plasma.
[0078] In some embodiments, a ratio of T to monosialylated ST of at
least two standard deviations less than a control ratio of T to ST
indicates that the first dosage of the therapeutic agent is
effective for the treatment of the hyposialylation disorder and/or
that the therapeutic agent has not been administered for a
sufficient duration of time to treat the subject. In yet other
embodiments, a ratio of T to monosialylated ST of at least three
standard deviations less than a control ratio of T to ST for a
control indicates that the first dosage of the therapeutic agent is
effective for the treatment of the hyposialylation disorder and/or
that the therapeutic agent has been administered for a sufficient
duration of time to treat the subject. In further embodiments, the
control ratio is the mean ratio of T to ST in biological samples
from subjects that do not have the hyposialylation disorder. The
biological sample can be any biological sample of interest, such as
blood, an extract from a biopsy, such as an extract of platelets,
white blood cell, red blood cells, kidney cells, muscle cells,
heart cells, brain cells, lung cells, or liver cells. The
biological sample can be urine or cerebrospinal fluid.
[0079] In certain aspects, these assays are performed at a
diagnostic laboratory, and the information is then provided to the
subject or a physician or other healthcare provider. In some
embodiments, the dosage of the therapeutic agent is decreased, and
a second lower dosage of the therapeutic agent is administered to
the subject. In additional embodiments, these methods can be used
to determine the lowest effective dosage of the therapeutic agent
of use to treat the subject. In yet other embodiments, the dosage
of the therapeutic is increased and administered to the subject. In
other examples, and additional dosage of the therapeutic agent is
administered to the subject.
[0080] Thus, in additional embodiments, the method can include
administering to the subject a second dosage of the therapeutic
agent, wherein the second dosage is the same, greater, or less than
the first dosage of the therapeutic agent. Monosialylated ST
antigen and T antigen are measured in a biological sample from the
subject, and the ratio of T to ST is determined.
[0081] In some embodiments, a ratio of T to monosialylated ST in
serum or plasma samples of less than about 0.0521, less than about
0.052, less than about 0.053, less than about 0.054, less than
about 0.055, less than about 0.056, less than about 0.057, less
than about 0.058, less than about 0.059, or less than about 0.06,
indicates that the second dosage of the therapeutic agent is
effective for the treatment of the hyposialylation disorder and/or
has been administered for a sufficient duration. In some
embodiments, a ratio of T to ST of less than about 0.05, about 0.04
or about 0.03 in the plasma or serum sample indicates that the
second dosage of the therapeutic agent is effective for the
treatment of the hyposialylation disorder and/or has been
administered for a sufficient duration. A ratio of T to ST of
greater than about 0.051, greater than about 0.052, greater than
about 0.053, greater than about 0.054, greater than about 0.055,
greater than about 0.056, greater than about 0.057 or greater,
greater than about 0.058, greater than about 0.059, or greater than
about 0.06 in the plasma or serum sample indicates that the second
dosage of the therapeutic agent is not effective for the treatment
of the hyposialylation disorder and/or has not been administered
for a sufficient duration. In other embodiments, a ratio of T to ST
of greater than about 07, about 0.08, about 0.09, or about 0.1 in
the plasma or the serum sample indicates that the second dosage of
the therapeutic agent is not effective for the treatment of the
hyposialylation disorder. Thus, in some embodiments, the methods
disclosed herein can be repeated to determine the lowest dosage of
an agent that is effective for the treatment of the subject.
Biological samples other than serum or plasma can also be used.
[0082] In some embodiments, in other samples than plasma or serum,
a ratio of T to monosialylated ST of at least two standard
deviations less than a control ratio of T to ST for a control
indicates that the second dosage of the therapeutic agent is
effective for the treatment of the hyposialylation disorder and/or
is administered for a sufficient duration to treat the subject. In
yet other embodiments, a ratio of T to monosialylated ST of at
least three standard deviations less than a control ratio of T to
monosialylated ST for a control indicates that the second dosage of
the therapeutic agent is effective for the treatment of the
hyposialylation disorder and/or that the therapeutic agent has been
administered for a sufficient duration of time to treat the
subject. In further embodiments, the control ratio is the mean
ratio of T to monosialylated ST in biological samples from subjects
that do not have the hyposialylation disorder. Thus, the methods
can be repeated to determine the lowest dosage of an agent that is
effective for the treatment of the subject. The biological sample
can be any biological sample of interest, such as an extract from a
tissue biopsy, such as an extract of platelets, white blood cell,
red blood cells, kidney cells, muscle cells, heart cells, brain
cells, lung cells, or liver cells. The biological sample can be
blood, urine or cerebrospinal fluid.
[0083] The methods can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more times to determine the lowest dosage of a therapeutic agent
that is effective for treating the subject, and/or the shortest
duration of administration that is effective for treating the
subject. The methods can also be used over the course of a
therapeutic regimen to monitor the efficacy of a therapeutic agent
for the treatment of the subject.
[0084] The disclosed methods can include comparing the ratio of T
to monosialylated ST to a control. The control can be a standard
value, or the ratio of T to monosialylated ST in a biological
sample from a subject known not to have the sialylation disorder,
such as the hyposialylation disorder.
[0085] For any and all of the methods disclosed herein, the
hyposialylation disorder can be any hyposialylation disorder of
interest. In other non-limiting examples, the hyposialylation
disorder is a congenital disorder of glycosylation, such as an
N-linked glycosylation disorder, O-linked glycosylation disorder,
multiple glycosylation disorder or disorder of glycolipid
synthesis. In further non-limiting examples, the hyposialylation
disorder is not a congenital disorder of glycosylation, such as a
CDGI or CDGII disorder. Hyposialylation disorders include, but are
not limited to hereditary inclusion body myopathy, also called GNE
myopathy or distal myopathy with rimmed vacuoles (DMRV). In some
embodiments, the hyposialylation disorder can include renal
hyposialylation. These include, but are not limited to, minimal
change nephrosis, lupus nephritis, and IgA nephropathy. In other
embodiments, the hyposialylation disorder is a sleep disorder,
including those with reduced REM sleep, or a disorder due to brain
hypofunction, see U.S. Published Patent Application No.
2011/0212917, incorporated herein by reference. In additional
embodiments, the hyposialylation disorder is a neurodegenerative
disorder, such as a disorder that includes accumulation of
.beta.-amyloid, such as Alzheimer's disease and Lewy body dementia.
In additional examples, the neurodegenerative disorder is a
cognitive disorder involving brain hypofunction. In other
embodiments, the hyposialylation disorder is a liver disorder, or a
muscular disorder. In some specific, non-limiting examples, the
hyposialylation disorder is a congenital muscular dystrophy or
inclusion body myositis. The hyposialylation disorder can be a
cancer. In further embodiment, the hyposialylation disorder is a
kidney or a liver disorder, such as podocytopathies, minimal change
nephrosis, focal segmental glomerulosclerosis, membranous
glomerulonephritis, and other forms of unexplained idiopathic
nephrotic syndrome, glomerular basement membrane diseases (such as
Alport disease and thin membrane disease). In some examples, the
kidney disorder is characterized by segmental splitting of the
glomerular basement membrane and/or podocytopathy due to disturbed
polyanions on podocyte membranes In an additional embodiment, the
sialylation disorder is glomerular nephropathy. In further
embodiments the sialylation disorder is diabetic nephropathy. In
yet other embodiments, the hyposialylation disorder is a congenital
disorder of glycosylation such as galactosemia, deficiency in
Conserved Oligomeric Golgi Complexes (COG), such as COG4 or COG7, a
Deficiency of Transmembrane Protein 165 (TEM 165),
Posphoglucomutase 1 (PGM1) Deficiency or Posphoglucomutase 3 (PGM3)
Deficiency. The method disclosed herein can be used to confirm the
diagnosis of these disorders. Thus, in some embodiments, a subject
is selected suspected of having one of these disorders, such as due
to clinical symptoms. The methods disclosed herein can be used to
determine the effectiveness of a therapeutic agent for treating one
of these disorders in a subject of interest.
[0086] The method can include purifying O-glycans from the
biological sample. Thus, the method can include releasing
O-glycans, such as by treating the biological sample with sodium
hydroxide and sodium borohydrate. Suitable concentrations of sodium
hydroxide and sodium borohydrate are, for example, about 1M sodium
borohydrate in 0.05M sodium hydroxide. In some embodiments,
O-glycans are purified from the biological sample. Methods for
purifying O-glycans include organic solvent extraction with
methanol, and ion-exchange chromatography, such as with AG 50W-X8
resin (Bio-Rad, Hercules, Calif.). Exemplary non-limiting methods
are disclosed in the examples section.
[0087] Disclosed herein are methods of detecting biomarkers for
hyposialylation disorders in order to detect the hyposialylation
disorder or to determine if a therapeutic agent is effective for
the treatment of this disorder, and methods for detecting a
hyposialylation disorder. The monosialylated ST antigen and T
antigen biomarkers may be detected using any means known to those
of skill in the art, including the use of antibodies that
specifically bind T antigen, antibodies that specifically bind ST
antigen (see, for example, Cao et al. Histochem. Cell Biol. 106,
197-207 (1996)), and/or the use of lectins that bind T and/or ST
antigen, see for example, Almogren et al. Front Biosc S4: 840-863
(2012), incorporated herein by reference. These methods include
fluorescence activated cell sorting (FACS) and enzyme linked
immunosorbent assays (ELISA), Western blotting and 2D gel
electrophoresis. These methods can utilize both lectins and
antibodies; suitable antibodies are disclosed, for example, in
Published U.S. Patent Application No. 2012/0294859. In some
embodiments, these methods are used to detect monosialylated ST
antigen and T antigen on white blood cells, platelets, red blood
cells, or other tissues. Generally, the monosialylated ST antigen
and T antigen biomarkers are quantitated.
[0088] In particular disclosed embodiments of the method, the
biomarkers are detected as a ratio using mass spectrometry. Any
mass spectrometry technique known to those of ordinary skill in the
art to be suitable for analyzing biological molecules can be
utilized. For example, mass spectrometric techniques contemplated
herein include mass spectrometry techniques using various
ionization techniques (such as, but not limited to, matrix-assisted
laser desorption/ionization (MALDI), electrospray, thermospray, and
the like) coupled with one or more mass analyzer components (such
as, but not limited to, time-of-flight 66BTOF], quadrupole, and ion
traps). Any of the mass spectrometry detection methods used herein
may also be modified to perform tandem mass spectrometry, and/or
may be modified to employ additional analytical techniques, such as
liquid chromatography, gas chromatography, and ion mobility.
[0089] Mass spectrometry has been used as a powerful tool to
characterize polymers such as glycans because of its accuracy
(.+-.1 Dalton) in reporting the masses of fragments generated
(e.g., by enzymatic cleavage), and also because only pM sample
concentrations are required. For example, matrix-assisted laser
desorption ionization mass spectrometry (MALDI-MS) has been
described for identifying the molecular weight of polysaccharide
fragments in publications such as Rhomberg et al. PNAS USA 95,
4176-4181 (1998); Rhomberg et al. PNAS USA 95, 12232-12237 (1998);
and Ernst et al. PNAS USA 95, 4182-4187 (1998). Other types of mass
spectrometry known the art, such as electron spray-MS, fast atom
bombardment mass spectrometry (FAB-MS) and collision-activated
dissociation mass spectrometry (CAD) can also be used. However, the
disclosed methods are not limited to the use of mass spectrometry.
Other methods of use include, but are not limited to, capillary
electrophoresis (CE), NMR, and HPLC with fluorescence
detection.
[0090] The techniques, including mass spectrometry techniques
disclosed herein may be used to determine the ratio of biomarkers
present in a biological sample. For example, particular embodiments
concern the corel monosialylated ST antigen and the T antigen. The
ratio of these two antigens within a particular biological sample
may be determined by using the disclosed mass spectrometry
techniques to produce one or more ions identifying the particular
antigen. For example, a sample may be added to a mass spectrometer,
which promotes fragmentation of the components within the sample to
produce various different ions associated with each component.
[0091] Multiple reaction monitoring may be used to produce a unique
fragment ion that can be monitored and quantified. In particular
disclosed embodiments, the parent mass of the compound is specified
and the sample comprising the compound is monitored for the unique
fragment ion. Typically, the parent mass/ion of the compound is
selected and fragmented and either a particular fragment, the
unique fragment ion, is analyzed or all fragments from the parent
ion are analyzed. The ratio of each compound can be determined
using quantitative mass spectrometry, such as by using an internal
standard. In particular disclosed embodiments, the monosialylated
ST and T antigen have different mass transitions, which can be
determined in order to quantify the ratio of the two antigens in a
biological sample. Typically, the monosialylated ST antigen will
have a parent mass (or parent ion m/z) of 895 and the fragment ion
is 520. The T antigen can have a parent mass (or parent ion m/z) of
534 and the fragment ion is 298. The concentration of each of the
monosialylated antigen and the T antigen can be measured by
comparing the signals from the internal standard with that produced
by either the ST or T antigens. In particular disclosed
embodiments, one or more calibration curves may be produced using
various different concentrations of either antigen.
[0092] According to one embodiment of the disclosed methods, a
biological sample (e.g., a blood sample, plasma sample, tissue
extract etc.) is collected and prepared for analysis. As an
example, an internal standard may be added to the biological sample
in solution (e.g., aqueous solution). The biological sample may
then be treated with a buffered base solution (e.g., an aqueous
solution of sodium borate and sodium hydroxide) in order to promote
denaturation of the serum proteins. The solution may be neutralized
using an appropriate neutralizing solution (e.g., acetic acid in
methanol), and the desired glycans extracted using methanol. The
extracted glycans may be desalted using an ion exchange resin and
then dried.
[0093] Once the desired biological sample is obtained, it may be
manipulated in order to promote analysis using the disclosed mass
spectrometric method. In particular disclosed embodiments, desalted
glycans may be permethylated using a base and appropriate
methylating agent. Solely by way of example, the glycan may be
exposed to an aqueous solution of sodium hydroxide in
dimethylsulfoxide (DMSO) and then treated with methyl iodide. After
extraction, the permethylated glycans are purified, such as by a
SPE C18 column, and used in the disclosed mass spectrometric
analysis.
[0094] According to one embodiment, the permethylated glycans are
analyzed using tandem mass spectrometry coupled with
high-performance liquid chromatography (HPLC-MS/MS); however, any
suitable mass spectrometric methods may be used as disclosed
herein. In particular disclosed embodiments, a suitable
buffer/solvent system is selected for the HPLC analysis portion of
the analytical technique. For example, a two-buffer system may be
used. Particular disclosed embodiments concern using a first buffer
of acetonitrile/formic acid/water having ratios of 1:0/1:99
(v:v:v), respectively, and a second buffer of acetonitrile/formic
acid/water having ratios of 99:0/1:1 (v:v:v), respectively.
Exemplary flow rate protocols are provided herein. In particular
disclosed embodiments, mass spectrometry analysis is conducted
using an enhanced product ion source in the positive mode and one
or more quadrupole mass analyzers. Exemplary non-limiting methods
are disclosed in the Examples section below.
Exemplary Therapeutic Agents
[0095] Methods are disclosed herein for determining the
effectiveness of a therapeutic agent for treatment of a
hyposialylation disorder in a subject, and/or that include
administering a therapeutic agent to a subject. The therapeutic
agent can be any agent of interest, including, but not limited to,
N-acetyl-D-mannosamine (ManNAc), mannosamine, N-acetylneuraminic
acid (Neu5Ac), another sialic acid, or one or more sialylated
compounds. The one or more sialylated compounds can be the one or
more sialylated compound comprises intravenous immunoglobulin (WIG)
or sialyllactose. The therapeutic agent can be mannose or a
derivative thereof, such as a mannose 1-phosphate derivative as
disclosed, for example, in European Patent No. EP 1521761, which is
incorporated herein by reference.
[0096] The structure of N-acetylneuraminic acid (Neu5Ac) is shown
below.
##STR00003##
[0097] Intravenous immunoglobulin is pooled, polyvalent
immunoglobulin G (IgG) extracted from donors. In some embodiments,
IVIG is administered at a high dosage, such as about 100 to 400 mg
per kg of body weight, or about 1 to about 2 grams WIG per kg body
weight.
[0098] N-acetylmannosamine and derivatives thereof are useful for
treating a variety of diseases and conditions, see for example,
U.S. Published Patent Application No. 2013/0058998-A1, and U.S.
Pat. No. 8,410,063, both incorporated herein by reference.
N-acetyl-D-mannosamine is an uncharged, key compound in the Neu5Ac
acid biosynthetic pathway. Neu5Ac is the most abundant mammalian
sialic acid, and is a precursor of most sialic acids. In
particular, there is a regulated, rate-limiting enzymatic step in
the pathway that leads to Neu5Ac formation, and this rate-limiting
step gives rise to N-acetyl-D-mannosamine. Once
N-acetyl-D-mannosamine is formed or administered, no other
enzymatic step leading to the formation of Neu5Ac is subject to
feedback inhibition. Administration of N-acetyl-D-mannosamine leads
to increased amounts of Neu5Ac. The structure of
N-acetylmannosamine is shown below.
##STR00004##
N-acetylmannosamine and derivatives thereof can also be used for
the treatment of hyposialylation disorders. Structures of such
N-acetylmannosamine derivatives are provided in Formula I.
##STR00005##
wherein:
[0099] R.sub.1, R.sub.3, R.sub.4, or R.sub.5 is hydrogen, lower
alkanoyl, carboxylate or lower alkyl; and
[0100] R.sub.2 is lower alkyl, lower alkanoylalkyl, lower alkyl
alkanoyloxy.
Alkyl, alkoxy, alkenyl, and alkynyl denote both straight and
branched groups; but reference to an individual radical such as
"propyl" embraces only the straight chain radical, a branched chain
isomer such as "isopropyl" being specifically referred to. Lower
alkyl refers to (C.sub.1-C.sub.6)alkyl. Such a lower alkyl or
(C.sub.1-C.sub.6)alkyl can be methyl, ethyl, propyl, isopropyl,
butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;
(C.sub.3-C.sub.6)cycloalkyl can be cyclopropyl, cyclobutyl,
cyclopentyl, or cyclohexyl;
(C.sub.3-C.sub.6)cycloalkyl(C.sub.1-C.sub.6)alkyl can be
cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,
cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl,
2-cyclopentylethyl, or 2-cyclohexylethyl; (C.sub.1-C.sub.6)alkoxy
can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy,
sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;
(C.sub.2-C.sub.6)alkenyl can be vinyl, allyl, 1-propenyl,
2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl,
2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl,
3-hexenyl, 4-hexenyl, or 5-hexenyl; (C.sub.2-C.sub.6)alkynyl can be
ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl,
1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl,
2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;
(C.sub.1-C.sub.6)alkanoyl can be acetyl, propanoyl or butanoyl;
halo(C.sub.1-C.sub.6)alkyl can be iodomethyl, bromomethyl,
chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl,
2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl;
hydroxy(C.sub.1-C.sub.6)alkyl can be hydroxymethyl, 1-hydroxyethyl,
2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl,
1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl,
1-hydroxyhexyl, or 6-hydroxyhexyl; (C.sub.1-C.sub.6)alkoxycarbonyl
can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,
isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or
hexyloxycarbonyl; (C.sub.1-C.sub.6)alkylthio can be methylthio,
ethylthio, propylthio, isopropylthio, butylthio, isobutylthio,
pentylthio, or hexylthio; (C.sub.2-C.sub.6)alkanoyloxy can be
acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy,
or hexanoyloxy.
[0101] Administration of N-acetylmannosamine and/or derivatives
thereof can lead to a reduction in proteinuria (e.g., lower amounts
of protein in the urine), a reduction in hematuria (e.g., lower
amounts of red blood cells in the urine) and improvement of muscle
function (e.g., in patients with muscular atrophy). Effective
amounts for human patients are, for example, about 0.01 g/day to 50
g/day, about 0.1 g/day to about 50 g/day, of about 0.2 g/day to
about 25 g/day, from about 0.3 g/day to about 12 g/day, from about
0.4 g/day to about 10 g/day, from about 0.5 g/day to about 8 g/day,
and from about 0.7 g/day to about 6 g/day. N-acetylmannosamine
and/or derivatives thereof may be administered as single or divided
dosages, for example, of at least about 0.01 mg/kg to about 500 to
750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg,
at least about 0.1 mg/kg to about 200 to 400 mg/kg or at least
about 1 mg/kg to about 25 to 200 mg/kg of body weight, although
other dosages may provide beneficial results. Generally,
N-acetylmannosamine and/or a derivative thereof is administered for
periods of time sufficient to increase the amount of sialic acid
(Neu5Ac) in the mammal and thereby achieve a therapeutic benefit.
The use of N-acetylmannosamine is disclosed in PCT Publication No.
WO 2008/150477, which is incorporated herein by reference.
[0102] Exemplary components of the sialic acid (e.g., Meu5Ac)
biosynthesis pathway can be used as therapeutic agents, and include
mannosamine, N-acetyl mannosamine (ManNAc) (see above),
ManNAc-6-phosphate (ManNAc-6-P), UDP-GlcNAc, N-acetylneuraminic
acid (Neu5Ac), NeuAc-9-phosphate (NeuAc-9-P), sialic acid (i.e.,
5-N-acetylneuraminic acid and derivatives), and CMP-Neu5Ac. Hence,
certain treatments include the direct administration of one or more
of these components as compounds, or as derivatives or
pharmaceutically acceptable salts thereof, including extended
release formulations of such compounds (see, e.g., PCT Application
No. PCT/US2011/043910, and U.S. Published Patent Application No.
2013/0058998A1, each of which is incorporated by reference in its
entirety) or encapsulated compounds. In some examples, these
compounds can be formulated for release over a defined time period
such as 12, 24, 48, or 72 hours. The term "derivative" encompasses
derivatives, analogs, prodrugs, and unnatural precursors of a given
compound. In specific embodiments, the compound in the Neu5Ac
biosynthesis pathway or derivatives thereof that do not include
glucose, or a pharmaceutically acceptable salt thereof.
[0103] Therapy with nucleic acid can also be utilized. Any gene
involved in the sialic acid biosynthesis pathway, such as a gene
involved in the Neu5Ac pathway, can be utilized. In some
embodiments, methods for increasing Neu5Ac/sialic acid production
by providing a subject with a wild-type GNE-encoding nucleic acid
sequence that is optionally operably linked to a regulatory
element, such as a promoter and/or enhancer sequence (see U.S.
Application No. 2011/027373; WO 2008/097623; and U.S. Application
No. 2009/029811, which are incorporated by reference in their
entireties). This gene replacement therapy targets GNE, which is
defective in GNE myopathy (HIBM) patients, typically due to an
autosomal recessive mutation of the GNE gene (see, e.g., Nemunaitis
et al. J Gene Med 12, 403-12 (2010)). The GNE gene encodes the
bi-functional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase. Thus, in
some embodiments, therapy includes gene replacement therapy with
wild type or modified GNE gene, genes involved in the sialic acid
synthesis pathway, or other genes.
[0104] The appropriate dosage of any of these therapeutic agents,
or any other agent of use to treat a hyposialylation disorder, can
be determined using methods disclosed herein.
[0105] The disclosure is illustrated by the following non-limiting
Examples.
EXAMPLES
Example 1
Materials and Methods
[0106] Materials:
[0107] Iodomethane, dimethyl sulfoxide anhydrous (DMSO),
2,5-dihydroxybenzoic acid (DHB), sodium hydroxide, trifluoroacetic
acid (TFA), raffinose, sodium borohydrate, and sodium acetate were
obtained from Sigma-Aldrich (St. Louis, Mo.). PNGase F, including
denaturing buffer, digestion buffer and NP-40 were obtained from
New England Biolabs (NEB, Ipswich, Mass.). Extra-Clean SPE
Carbograph columns were obtained from Grace Davison Discovery
Sciences (Deerfield, Ill.). Sep-Pak Vac 3cc C18 cartridges were
from Waters (Milford, Mass.). p-lacto-N-hexaose (pLNH) was obtained
from V-labs (Covington, La.). Methanol, chloroform and acetonitrile
were from Fisher Scientific (Fair Lawn, N.J.).
[0108] Samples:
[0109] Normal serum or plasma samples were obtained from the
collection at the Emory Clinical Biochemical Genetics Laboratory. A
total of 150 serum or plasma specimens from normal population were
used to collect the reference range for the N-glycans. Forty normal
control serum or plasma was analyzed for the reference range of the
O-glycans. The de-identified 6 sera from previously characterized
patients with different CDG-II disorders were provided by Dr.
Hudson Freeze (Sanford-Burnham Medical Research Institute, La
Jolla, Calif.) and included COG4 (Ng et al. Mol Genet Metab 102,
364-367(2011)), COG 7 (Wu et al. Nat Med 10, 518-523 (2004)),
PGM1-CDG (mixed CDG-I and II with deficiency in nucleotide sugar
metabolism) (Perez et al. J Inher Metab Dis 36, 535-542 (2013)),
TMEM165-CDG (Foulquier et al. Am J Hum Genet, 91, 15-26 (2012)),
and one CDG-IIx with mixed CDG-I and II (Mandato et al. Pediatr Res
59, 293-298 (2006)).
[0110] Sample Preparation for N-Glycan and O-Glycans:
[0111] Sample preparations for N- and O-glycans were carried out by
PNGaseF digestion and .beta.-elimination respectively as described
before (Liu et al. Mol Genet Metab 106, 442-454 (2012)). 20 .mu.L
serum or plasma and 150 pmols of internal standard pLNH were used
for N-glycan preparation and 10 .mu.L serum or plasma and 1,250
pmols of internal standard raffinose was used for O-glycan
preparation. All the purified glycans were lyophilized overnight to
complete dryness.
[0112] Permethylation:
[0113] Both N- and O-glycans were permethylated as previously
described with minor modifications (Faid et al. Proteomics 7,
1800-1813 (2007)). Briefly, a slurry of DMSO/NaOH was freshly
prepared (0.4 ml) and was added to the dried glycan sample with 0.1
ml of iodomethane and mixed thoroughly for 60 min at room
temperature. The permethylation reaction was then quenched by
addition of 0.5 ml water and glycans were extracted by Chloroform
(0.5 ml) and washed by water (0.5 ml) for 4 times and was then
dried. The dried sample was further purified by the SeP-Pak C18
column and then lyophilized overnight and ready for analysis by
mass spectrometry.
[0114] N-Glycan and O-Glycan Profile Analysis by MALDI-TOF/TOF:
[0115] The permethylated N- or O-glycans were analyzed on an
Applied Biosystem MALDI-TOF/TOF 4800 plus (Applied Biosystems,
Foster City, Calif.) as described before (Liu et al. Mol Genet
Metab, 106, 442-454 (2012)). 11 mg/ml 2,5-dihydroxybenzoic acid
(DHBA) and 1 mM sodium acetate in 50% methanol was used as matrix
buffer.
[0116] LC-MS/MS Conditions for Mucin Core 1 T-Antigen and
Monosialyl ST-Antigen Quantification:
[0117] HPLC Separation of the small O-glycans released by
.beta.-elimination comprised of the core 1 disaccharide
Gal.beta.1-3GalNAc (T-antigen) and monosialyl-antigen (ST-antigen)
was achieved with a Shimadzu Prominence 20AD LC and a Thermo gold
3-.mu.m C18 column (2.0.times.100 mm) as described previously (Liu
et al. Mol Genet Metab 106, 442-454 (2012)).
[0118] Preparation of Isotope Labeled Standards:
[0119] The purified milk sugar pLNH was labeled in the
permethylation step as described above with either .sup.12C or
.sup.13C by using either iodomethane-.sup.12C or
iodomethane-.sup.13C.
Example 2
Sample Stability
[0120] The stability of the glycans in serum and plasma was tested
by storing three aliquots of serum or whole blood (plasma) from the
same donor at room temperature (RT) for 0 hour (hr), 24 hr and 48
hr (Table A & B, below). Comparison at different time points
demonstrated that the relative abundance of major N- and O-glycans
in human serum or plasma was stable at RT for at least 48 hours,
thus it is generally feasible to ship sera or whole blood at RT for
glycan profiling tests. The percentage of CVs between relative
abundance of N-glycans from these samples was less than 20%, and
there was also no significance difference observed between the
human serum and the human plasma (Tables A & B).
TABLE-US-00001 TABLE A The Stability of N-Glycans of Total Plasma
and Serum Glycoproteins at Room Temperature N- linked Glycans
Plasma (% total glycan) Serum(% total glycan) (m/z) 0 hr 24 hr 48
hr 0 hr 24 hr 48 hr Mean SD CV % 1579.8 0.98 0.90 0.91 1.23 0.73
1.24 1.00 0.20 20 1661.8 0.14 0.13 0.12 0.16 0.12 0.17 0.14 0.02 14
1783.9 0.84 0.78 0.78 1.19 0.68 1.02 0.88 0.19 21 1835.9 0.10 0.06
0.10 0.09 0.06 0.09 0.08 0.02 21 1865.9 0.30 0.35 0.31 0.33 0.28
0.34 0.32 0.02 8 1982 0.40 0.45 0.42 0.51 0.33 0.42 0.42 0.06 14
1988 0.24 0.28 0.22 0.36 0.25 0.29 0.27 0.05 18 2156.1 0.09 0.12
0.12 0.10 0.10 0.15 0.11 0.02 18 2192.1 0.37 0.35 0.33 0.51 0.34
0.32 0.37 0.07 20 2227.1 0.89 0.94 0.92 0.75 0.78 0.81 0.85 0.08 9
2285.2 0.12 0.12 0.15 0.05 0.11 0.07 0.10 0.03 34 2396.2 0.26 0.47
0.65 0.74 0.50 0.52 0.52 0.16 31 2431.2 3.81 3.90 4.26 3.64 3.37
3.29 3.71 0.36 10 2605.3 0.79 0.72 0.90 0.81 0.85 0.69 0.79 0.08 10
2792.4 13.18 14.61 15.16 14.38 14.96 12.46 14.12 1.07 8 2966.5 1.34
1.60 1.65 1.56 1.67 1.33 1.52 0.15 10 3241.6 0.35 0.48 0.51 0.34
0.47 0.40 0.42 0.07 17 3415.7 0.18 0.13 0.15 0.21 0.25 0.11 0.17
0.05 31 3602.8 1.41 1.59 2.09 1.44 1.82 1.16 1.59 0.33 21 3776.9
1.01 1.00 1.52 0.88 1.25 0.80 1.07 0.27 25
TABLE-US-00002 TABLE B The Stability of O-Glycan of Total Plasma
and Serum Glycoprotein at Room temperature O- linked Plasma (uM)
Serum (uM) Glycans 0 hr 24 Hr 48 hr 0 hr 24 hr 48 hr Mean SD CV %
T- 0.80 0.89 0.89 0.91 0.86 0.83 0.86 0.04 0.13 antigen Monosialy
18.45 18.05 18.65 17.7 18.4 17.4 18.11 0.48 10.4 T antigen
Example 3
Precision and Recovery
[0121] The consistency of permethylation and the recovery of
glycans during the glycans purification steps were evaluated using
isotope labeled standards. First, the light (.sup.12C) and heavy
(.sup.13C) isotope-labeled internal standards were permethylated as
described above. Equal aliquots of light- and heavy-labeled pLNH
were prepared separately and mixed together and analyzed by
MALDI-TOF. The ratio of peak areas of light- and heavy-labeled
standards was 1.02 and the partial permethylated pLNH was <5%,
indicating that the permethylation reactions were >95% complete
and there was minimal variation in permethylation. Thus, the
measured intensity of permethylated standard accurately reflects
the molar ratio of starting material. Next, the unlabeled pLNH 500
pmol was spiked into the plasma and carried through the whole
process. After these final steps, 500 pmols .sup.13C-labeled
standard were added to the mixture before it was analyzed by
MALDI-TOF. The ratio of unlabeled standard (m/z 1375) and peak area
of .sup.13C-labeled standard (m/z 1395) was 0.97, and the recovery
of pLNH through purification was estimated at 97%. The high
recovery rate indicates that the purification steps are very
efficient and monitoring the signal of known amount of internal
standard should be sufficient to monitor the efficiency of these
steps. The fragmentation pattern of the glycans were obtained using
MALDI-TOF/TOF mode for additional information on sugar component
and structure.
[0122] The variations of interday and intraday runs of O-glycan and
N-glycan analysis were measured to evaluate the test precision and
reproducibility. Intraday CVs of the four most abundant N-glycans,
T-antigen and ST-antigens were less than 20%. Interday CVs were
also less than 20% (Table 1).
TABLE-US-00003 TABLE 1 Precision of N- and O-Glycan Analysis by
MALDI-TOF in Control Plasma Inter-assay (n = 20) Intra-assay (n =
10) Median % Median % Structure N-glycan m/z (%) SD CV (%) SD CV
##STR00006## Neu5Ac2Hex5HexNAc4 (Disialo biantennary) 2792.4 15.7
1.8 11 15.7 1.0 6 ##STR00007## Neu5Ac2Fuc1Hex5Hex NAc4 (Disialo
biantennary fucosylated) 2966.5 1.6 0.2 11 1.2 0.1 9 ##STR00008##
Neu5Ac2Hex6HexNAc5 (Disialo triantennary) 3241.6 0.7 0.1 20 0.7 0.1
17 ##STR00009## Neu5Ac3Hex6HexNAc5 (Trisialo triantennary) 3602.8
2.4 0.3 12 2.6 0.4 18 Inter-assay (n = 20) Intra-assay (n = 10)
Median % Median % Structure O-glycan MRM (.mu.M) SD CV (.mu.M) SD
CV ##STR00010## T-antigen 534/298 0.74 0.08 11 0.73 0.06 7.8
##STR00011## Monosialylated-T- antigen 895/520 19.9 3.4 17 23.2 1.4
6.0 : GlcNAc, : Man, : Gal, : NeuNAc, : Fuc
Example 4
O-Glycan Profiles of Total Serum or Plasma Total Glycoproteins
[0123] O-glycan profiles of 40 normal plasma or serum samples were
analyzed, along with 6 CDG-II samples. A small amount of T-antigen
was spiked into a normal plasma sample and was run as a positive
control with each O-glycan analysis to help identify all the major
O-glycan species shown in FIG. 1A, including T-antigen (m/z 534),
monosialyl ST-antigen (ST) (m/z 895), disialyl-T-antigen (m/z
1256), monosialyl core 2 (m/z 1344) and disialyl core 2 (m/z 1705),
with ST-antigen at m/z 895 as the most abundant species. The
relative quantity of ST-antigen and disialyl-T-antigen were
estimated by comparing their intensity to the internal standard
(raffinose at m/z 681). A clear reduction of abundance could be
detected by semi-quantifying monosialyl ST-antigen and
disialyl-ST-antigen in 3 COG patients (red circles) comparing to
controls (blue triangles) with MALDI-TOF analysis (FIG. 1B) (see
Faid et al. Proteomics 7, 1800-1813 (2007)). However, the abundance
of T-antigen at m/z 534 is difficult to evaluate by MALDI-TOF.
[0124] In order to achieve better quantification of serum or plasma
O-glycans, a LC-MS/MS method was developed to quantify T-antigen
and monosialylated ST-antigen. The chromatograms of extracted
multiple reaction monitoring (MRM) for T-antigen, and
monosialylated ST-antigen are shown in FIG. 2A. The specificities
of the MRM transitions for each glycan are supported by
MRM-triggered enhanced parent ion (EPI) profiles for each O-glycan
shown in FIG. 2B. Pure T-antigen standard was run to evaluate the
recovery, limit of quantification and linearity of the LC-MSMS
analysis (FIG. 2C). The analytical measurement range of T-antigen
was 0.0625-5 .mu.M and recovery was >90%. The ratio between T-
and monosialylated ST-antigens was also obtained to evaluate the
sialylation of core 1 O-glycans. The reference ranges of both T-
and monosialylated ST-antigens, along with the T- and
monosialylated ST-antigen ratio, were obtained from 40 normal
control sera or plasma. All six positive CDG samples have
relatively high T-antigen and low monosialylated ST-antigen (FIG.
2D). The distinction between affected patients and control was best
achieved by measuring T/monosialylated ST-antigen ratio. The T/ST
ratio of three known COG patients were at 0.17(COG4), 0.11 (COG7,
patient 1), 0.11 (COG7, patient 2) with the reference interval of
the T/ST-antigen ratio from controls at 0-0.056. All three known
COG samples had low monosialylated ST-antigen levels, and increased
T/ST-antigen ratio. However, an increased T-antigen level was
detected in the COG4-CDG patient, but not in two COG7-CDG samples
(Table 2).
TABLE-US-00004 TABLE 2 Quantification of Serum O-glycans in
Patients with CDG-II and Normal Controls O-glycan T(.mu.M)
Monosialyl-T(.mu.M) T/Monosialyl-T ratio Control <=1.03 >=13
<=0.058 TMEM165 1.67 9.45 0.177 PGM1 (mixed 1.44 9.38 0.154 type
I and II) COG4 1.23 6.95 0.176 COG7 p1 0.80 7.67 0.105 COG7 p2 0.73
6.33 0.116 Mixed type 1.30 19.02 0.068 I and II
[0125] Without being bound by theory, relatively low T-antigen in
these two samples from patients with COG7 deficiency could reflect
more severely affected Golgi protein transporting and more severe
clinical phenotype in these patients (Wu et al. Nat Med 10, 518-523
(2004)). Increased T/ST-antigen ratios were detected in PGM1-CDG,
which is similar to what was reported in galactosemia (Liu et al.
Mol Genet Metab 106, 442-454 (2012)). O-glycan quantification in
TMEM165-CDG showed significant increase of T-antigen, mildly
reduced monosialylated ST-antigen, and increased T/ST-antigen
ratio, providing evidence that TMEM165-CDG is a multiple
glycosylation disorder (Table 2). Increased T/ST-antigen ratio was
found, reflecting mild undersialylation of O-linked protein
glycosylation in the serum from a known patient with CDG-IIx.
Without being bound by theory, the finding of undersialylation of
O-glycans and possible deficiencies of multiple Golgi
glycosyltransferases provides evidence that this new mixed type I
and type II is likely related to a deficiency in general Golgi
functions and affect multiple Golgi protein glycosylation
functions.
[0126] The abundance of T antigen and monosialylated ST-antigen in
the plasma or serum are of several orders of difference, and
MALDI-TOF analysis is very limited in quantifying T and ST. The
level of sialylation of O-glycans are reduced in almost all the
known multiple glycosylation disorders (Wu et al. Nat Med 10,
518-523 (2004); Mohamed et al. J Inherit Metab Dis 34, 907-916
(2011)). Therefore accurate quantification of T-antigen, the only
nonsialylated O-glycan detectable in plasma or serum glycoproteins,
is critical. Disclosed herein is an LC-MS/MS method to quantify
serum or plasma T- and ST-antigens. The disclosed methods provide
detection of mild undersialylation of O-glycans in total plasma or
serum glycoproteins, otherwise undetectable by MALDI-TOF analysis,
by utilizing the T/ST-antigen ratio. Using this LC-MS/MS method,
undersialylation of O-glycosylation was demonstrated in this
disorder. In the case of COG7 deficiency, both T-antigen and
monosialylated ST-antigen were reduced. The results show that the
reason that T-antigen appears increased in COG7-CDG by MALDI-TOF is
due to relative low intensity of monosialylated ST-antigen rather
than increased T-antigen.
[0127] It is estimated that 2-3% of the genome encodes for genes
important in glycosylation processes. A reliable clinical
biochemical screening test to accurately detect and profiling
CDG-IIx improves the diagnostic yield of CDG-IIx. The disclosed
methods provide a clear distinction between CDG positive samples
and the control population.
Example 5
T/ST Ratio in GNE Myopathy and Glomerulopathies
[0128] The following example shows that the ratio of plasma and/or
serum mucin core 1 T-antigen to monosialylated ST-antigen (T/ST
ratio) is an effective biomarker for diagnosis and prediction of
treatment outcome for GNE myopathy, and likely for other
hyposialylation disorders, such as kidney hyposialylation
disorders. For these studies, a cut-off value of 0.06 was used to
diagnose hyposialylation.
[0129] The quantification of the T/ST ratio was carried out using
the O-glycan quantification methods disclosed in Liu et al., Mol
Genet Metab 106, 442-454 (2012). Briefly, O-linked glycans from
human serum or plasma total glycoproteins were released by sodium
hydroxide with sodium borohydrate and desalted by ion-exchange
column and then permethylated before the quantity of T-antigen and
monosialyl ST-antigen was measured using LC-MS/MS method. Plasma
samples from 40 normal controls were used to establish the normal
range for the T/ST ratio at <0.056. A cut-off of 0.06 was used
to diagnose hyposialylation of O-linked protein glycosylation in
human plasma or serum.
[0130] Of the 38 tested plasma samples from patients with GNE
myopathy, 35 samples were in the abnormal T/ST ratio range (0.06 or
higher), indicating hyposialylation. Representative results are
shown in the tables below:
TABLE-US-00005 TABLE 3 ##STR00012## ##STR00013##
[0131] As shown in the above Table, even though individual
T-antigen or monosialylated ST-antigen values can be in the normal
range for a GNE myopathy patient, the T/ST ratio is in almost all
cases abnormal for subjects with GNE myopathy. Follow-ups with the
three patients whose samples were in the normal range revealed
that: (a) one patient (SIA79) was self-administering off-label
ManNAc, (b) one patient (Sia 14a before therapy and Sia14b the same
patient after therapy) had received IVIG therapy to increase
sialylation (before this therapy the T/ST ratio was 0.1, and after
the therapy it became 0.05), and (c) one patient (NH00016) had
received sialic acid one month before the test (her T/ST ratio was
0.05 after treatment, but after three months without medication,
her ratio increased to abnormal range of 0.7). These follow-ups
further show the sensitivity and reliability of this ratio in
diagnosing and monitoring hyposialylation, and determining the
effectiveness of therapeutic agents.
[0132] The table indicated plasma T/ST ratios in all patients and
serum T/ST ratios in some patients. The serum T/ST ratio is also
abnormal, indicating that patient's serum can also be used for the
T/ST biomarker.
[0133] Plasma or serum T/ST ratios from eighteen patients with
unexplained glomerulopathies (Renal 1-18 in Table 5) and from ten
patients with diabetic nephropathy (DN1-10 in Table 5) were also
tested.
[0134] In the unexplained glomerulopathy group, twelve patients had
ratios in the normal range (.ltoreq.0.06), and six patients had
ratios that indicated hyposialylation (>0.06). Kidney biopsies
were available from four patients from this group. Lectin
histochemistry on paraffin embedded slides of these kidney biopsies
(because lectins are sugar-binding protein they can help to
determine glomerular sialylation status, as described in Kakani et
al. Am J Pathol 180, 1431-40 (2012)) showed normal glomerular
sialylation in two patients (Renal 2 and Renal 5) with T/ST ratios
in the normal range; while glomerular hyposialylation was detected
in biopsy samples of two other patients (Renal 1 and Renal 3) which
had increased T/ST ratios (of 0.09 and 0.07, respectively). The
results are shown in the table below. Kidney biopsies of 40
patients with different glomerular diseases were also analyzed by
lectin staining (but no plasma or serum was available). Eight of
these were hyposialylated, indicating that glomerular
hyposialylation exists in this patient population. These results
suggest that the plasma or serum T/ST ratio can be a reliable
biomarker for detecting glomerular hyposialylation.
[0135] In the diabetic nepropathy group, all ten patients had T/ST
ratios in the abnormal range, there were no kidney biopsies
available of these ten patients. However, kidney biopsies of 15
other patients with diabetic nephropathy were analyzed by lectin
staining (but no plasma or serum was available from these
patients). Thirteen of these samples were hyposialylated,
indicating that glomerular hyposialylation occurs in (the majority
of) patients with diabetic nephropathy. These results again that
the plasma or serum T/ST ratio can be a reliable biomarker for
detecting glomerular hyposialylation in diabetic nephropathy.
TABLE-US-00006 TABLE 4 ##STR00014## ##STR00015##
All plasma samples from patients with diabetic nephropathy had a
T/ST ratio of greater than 0.052. The T/ST ratio was, therefore
greater than 0.06. In fact, for these patients, a T/ST ratio of
greater than 0.08 was observed.
Example 6
Additional Methods
[0136] Chemicals:
[0137] Iodomethane, dimethyl sulfoxide anhydrous (DMSO),
2,5-dihydroxybenzoic acid (DHB), sodium hydroxide, trifluoroacetic
acid (TFA), raffinose, sodium borohydrate, and sodium acetate were
all purchased form Sigma-Aldrich (St. Louis, Mo., USA).
N-Glycosidase F (PNGase F), including denaturation buffer,
digestion buffer, and NP-40 buffer were all purchased from New
England Biolabs (Ipswich, Mass.). Extra-Clean SPE Carbos were
purchased from Grace Davison Discovery Science (Deerfield, Ill.).
The Sep-Pak Vac C18 cartridge 3cc was from Waters (Milford, Mass.).
The P-Lacto-N-hexaose (pLNH) was from V-labs (Covington, La.).
Acetonitrile, chloroform, methanol, sodium hydroxide (w/w, 50%),
and sodium acetate were all from Fisher Scientific (Fairlawn, N.J.,
USA).
[0138] Preparation of O-Glycans for Analysis:
[0139] O-glycans were released from plasma glycoproteins and
prepared for analysis essentially as described by Carlson (J Biol
Chem 243: 616-626, 1968; Liu et al. Mol Genet Metab 2012, 106,
442-454 (2012)), with modifications as described. An internal
standard (1250 pmol raffinose in 5 .mu.L) was added to 10 .mu.L of
plasma and 65 .mu.l water for a final volume of 100 .mu.L. Next,
100 .mu.L of freshly prepared 2M sodium borate in 0.1M sodium
hydroxide was added to denature the serum proteins and release the
O-glycans; the mixture was incubated at 45.degree. C. for 16 hours
to ensure complete reaction. The reaction was neutralized by drop
wise addition of 1.6 mL of a 0.25M acetic acid-methanol solution,
and the O-glycans were extracted in methanol. Finally, the
extracted glycans were desalted through ion-exchange AG 50W-X8
resin (Bio-Rad, Hercules, Calif.) following the manufacture's
instruction and lyophilized overnight. The dried samples were
dissolved in DMSO for permethylation.
[0140] Permethylation:
[0141] Both N-glycans and O-glycans were permethylated as
previously described with minor modification (Guillard et al. Clin
Chem 57, 593-602 (2011)). Briefly, four NaOH pellets (approximately
375 mg) were crushed in 10 ml anhydrous DMSO, 0.5 .mu.L water, 0.4
ml of this slurry and 0.1 ml CH3I were added to the dried glycans
and the mixture was shaken vigorously for one hour. The mixture was
extracted five times sequentially with a mixture of 200 .mu.L water
and 600 .mu.L chloroform. Finally, the combined chloroform phases
were dried under nitrogen in the chemical hood (30 mins) and the
permethylated N- and O-glycans were resuspended in 50 .mu.L of 50%
methanol and further purified through a C18 Stage Tip (Thermo
Scientific, West Palm Beach, Fla.) as described.
[0142] Quantification of O-Linked Glycans Corel T-Antigen and
Sialyl ST-Antigen by Tandem Mass Spectrometry Coupled with
High-Performance Liquid Chromatography (HPLC-MS/MS):
[0143] HPLC separation of O-linked glycans was achieved with a
Shimadzu Prominence 20AD LC and a Thermo gold 3-.mu.m C18 column
(2.times.100 mm) The binary method used buffer A
(acetonitrile:formic acid: water; 1:0.1:99 (v:v:v)) and buffer B
(acetonitrile:formic acid: water; 99:0.1:1 (v:v:v)) with a flow
rate at 0.25 ml/min under the following gradient conditions: O-20
min, 50% to 80% buffer B; 20-28 min, 98% buffer B; 28-39 min, 50%
buffer B. An injection volume of 10 .mu.l was used for analysis of
each sample.
[0144] The API-QTRAP 5500 tandem mass spectrometry conditions were
as follows: ion source: EPI positive mode; curtain gas: 25; ion
source: 5500; source temperature: 600. MRM transitions for corel
T-antigen and monosialyl-T-antigen were: m/z 534/298 and m/z
895/520. The parent ion of the T antigen is 534, the parent ion of
the monosialylated ST antigen is 895, and the fragment ions are 298
and 520, respectively.
[0145] Calibration curves were constructed with 6 concentrations of
T-antigen (from 0.0625 to 5 .mu.M). The ST value is based on the
ratio of the ST over the T peak area, times the T absolute
value.
Example 7
Sialylation of Thomsen-Friedenreich Antigen is a Noninvasive
Blood-Based Biomarker for GNE Myopathy
[0146] GNE myopathy is an adult-onset progressive myopathy,
resulting from mutations in GNE, the key enzyme of sialic acid
synthesis. The exact pathomechanism of GNE myopathy is not known,
but likely involves aberrant sialylation. GNE myopathy muscle
biopsies demonstrated hyposialylation of O-linked glycans.
Therefore, the O-linked glycome of patients' plasma proteins was
analyzed using mass spectrometry. Most patients showed an increased
core 1 O-linked glycan, Thomsen-Friedenreich (T)-antigen, and/or
decreased amounts of its sialylated form, ST-antigen. Moreover, all
patients had increased ratios of T-antigen to ST-antigen compared
to unaffected individuals Importantly, the T/ST ratios were
normalized in a patient treated with intravenous immunoglobulins as
a source of sialic acid, indicating response to therapy. These
findings highlight plasma T/ST ratios as a robust blood-based
biomarker for GNE myopathy, and can help explain the pathology and
course of the disease.
Materials & Methods
[0147] Patients:
[0148] GNE myopathy patients were enrolled in either clinical
protocol NCT01417533, `A Natural History Study of Patients with
Hereditary Inclusion Body Myopathy`, or protocol NCT00369421,
`Diagnosis and Treatment of Inborn Errors of Metabolism and Other
Genetic Disorders.` Peripheral blood samples were obtained and used
for serum or plasma preparations.
[0149] Genomic DNA was isolated from white blood cell pellets, and
used for GNE mutation analysis for molecular validation of the GNE
myopathy diagnosis, as shown in the table below. Peripheral blood
from healthy donors without clinical complaints at the time of
donation were also obtained.
TABLE-US-00007 TABLE 5 Absolute T- and ST-Values (as visualized in
FIG. 6B) ##STR00016## ##STR00017## (normal ranges are highlighted
in grey)
[0150] Whole Blood Sample Preparations:
[0151] Serum (non-gel serum separator tube, clot activator) and
plasma (K2EDTA-anticoagulant) were isolated from whole blood using
standard protocols, followed by albumin and IgG depletion using a
Qproteome Albumin/IgG depletion kit (Qiagen). Protein purification
and concentration was performed with micron Ultra-0.5 mL
Centrifugal Filters (EMD Millipore, Billerica, Mass.). Selected
control samples were desialylated by incubation with 1 .mu.1(50U)
neuraminidase for 1 hour at 37.degree. C. (P0720, New England
Biolabs, Ipswich, Mass.). This neuraminidase (cloned from
Clostridium perfringens and overexpressed in E. coli) catalyzes the
hydrolysis of .alpha.2-3, .alpha.2-6, and .alpha.2-8 linked
N-acetyl-neuraminic acid residues from glycoconjugates.
[0152] Immunoblotting:
[0153] Serum (10-40 .mu.g) proteins were boiled at 95.degree. C.
for 5 min in Laemmli Sample buffer (Bio-Rad Laboratories) and
electrophoresed on 4-12% Tris-Glycine gels (Invitrogen), followed
by electroblotting onto nitrocellulose membranes (Invitrogen). The
membranes were incubates with Ponceau S red according to the
manufacturer's protocol (Sigma-Aldrich, St Louis, Mo.) to visualize
equal loading and transfer of proteins in each lane. The membranes
were either probed with primary antibodies against NCAM or with
different lectins. Two antibodies against NCAM were evaluated H-300
(sc-10735) and RNL-1 (sc-53007) (Santa Cruz Biotechnology, Santa
Cruz, Calif.), whose binding was visualized by IRDYEO 800CW
conjugated secondary anti-mouse (for RNL-1) or anti-rabbit (for
H-300) antibodies (LI-COR.RTM. Biosciences, Lincoln, Nebr., USA).
The antigen-antibody complexes were visualized with the LI-COW)
ODYSSEY.RTM. Infrared imaging system (LI-COR.RTM.Biosciences). For
lectin probing (FIG. 9A-9D), biotinylated SNA (Sambucus nigra
agglutinin) and WGA (wheat germ agglutinin) were purchased from
Vector Laboratories (Burlingame, Calif.), and biotinylated VVA
(Vicia villosa agglutinin) was purchased from EY Laboratories (San
Mateo, Calif.). IRDYE.RTM. 680Streptavidin (LI-COW) Biosciences,
Lincoln, Nebr.) was used to bind to biotin-labeled proteins and
visualized with a LI-COW) Odyssey Infrared imaging system (LI-COW)
Biosciences).
[0154] Muscle Lectin Histochemistry:
[0155] Paraffin embedded sections (5 .mu.m) were obtained from
control biceps muscle (National Disease Research Interchange
(NDRI), Philadelphia, Pa.), right gastrocnemius muscle from patient
GNE-21 (carrying GNE mutations D378Y and A631V), and left biceps
muscle from patient GNE-28 (carrying GNE mutations R129X and
V696M). The sections were deparaffinized in HEMO-DE.RTM.
(Scientific Safety Solvents, Keller, Tex.), rehydrated in a series
of ethanol solutions, followed by antigen retrieval (by microwaving
in 0.01M Sodium Citrate, pH 6.4) and blocking in Carbo-Free
Blocking solution (Vector Laboratories, Burlingame, Calif.). The
slides were incubated at 4.degree. C. overnight with each
fluorescein isothiocyanate (FITC)-labeled lectin aliquoted (5
.mu.g/mL) in CARBO-FREE.RTM.) blocking solution. The FITC-labeled
lectins VVA and WGA were purchased from purchased from EY
Laboratories (San Mateo, Calif.) and SNA was purchased from Vector
Laboratories (Burlingame, Calif.). After overnight incubation,
washes were performed with 0.1% Triton-X-100 in 1.times.
Tris-buffered saline (TBS). The lectin-stained slides were
incubated in 0.3% Sudan Black in 70% ethanol solution to reduce
autofluorescence. Slides were mounted with Vectashield containing
the nuclear dye DAPI (Vector Laboratories) and digitally imaged
with a Zeiss LSM 510 META confocal laser-scanning microscope (Carl
Zeiss, Microimaging Inc., Thornwood, N.Y.). Images were acquired
using a Plan-Apochromat 40.times. oil DIC objective. All images are
3D projections of confocal Z-stacks.
[0156] To verify lectin specificity (FIG. 8A-8B), each lectin was
incubated with its specific inhibitory carbohydrate for 1 hour
before overnight incubation on a slide. The inhibitory
carbohydrates used were Neu5Ac (Toronto Research Chemicals,
Toronto, Canada), for WGA and SNA, and GalNAc (Sigma-Aldrich) for
VVA. In addition, tissue slides were desialylated by incubation
with 5 .mu.l (50U) neuraminidase (P0720, New England Biolabs) for 1
hour at a 37.degree. C. in enzyme buffer.
[0157] Preparation and Permethylation of Plasma O-Linked Glycan
Species:
[0158] O-linked glycan species were released from total (not
albumin or IgG depleted) plasma or serum glycoproteins by
.beta.-elimination, essentially as described (Liu et al. Mol Genet
Metab 106, 442-454 (2012); Carlson et al. J Biol Chem 243, 616-626
(1968); Faid et al. Proteomics 7, 1800-1813 (2007); Xia et al. Anal
Biochem 442, 178-85 (2013)). Briefly, 10 .mu.L of plasma was mixed
with raffinose (1250 pmol in 5 .mu.L) internal standard and 65
.mu.l water for a final volume of 100 .mu.L. To denature the plasma
proteins and release the O-linked glycan species, the sample was
mixed with 100 .mu.L 2 M sodium borate in 0.1 M sodium hydroxide
(freshly prepared) and incubated at 45.degree. C. for 16 hours.
Next, 1.6 mL of 0.25 M acetic acid-methanol solution was drop wise
added to neutralize the reaction, followed by O-glycan extraction
with methanol. The extracted glycans were desalted through
ion-exchange AG 50W-X8 resin (Bio-Rad, Hercules, Calif.) and
lyophilized overnight.
[0159] For permethylation, four NaOH pellets (approximately 375 mg)
were crushed in 10 mL anhydrous dimethyl sulfoxide (DMSO) with 0.5
.mu.L water; 0.5 mL of this slurry and 0.2 mL CH3I were added to
the dried glycans and the mixture was shaken vigorously for 1 hour,
followed by five sequential chloroform/water (600 .mu.L/200 .mu.L)
extractions from which the chloroform fractions were pooled. These
combined chloroform phases were dried for 30 min under nitrogen (in
chemical hood) and the permethylated O-glycan species were
resuspended in 50 .mu.L of 50% methanol and further purified
through a C18 Stage Tip (Thermo Scientific, West Palm Beach, Fla.)
as described (Guillard et al. Clin Chem 57, 593-602 (2011)).
[0160] O-Linked Glycan Analysis by LC-MS/MS and MALDI-TOF/TOF:
[0161] High performance liquid chromatography (HPLC) separation
coupled with an electrospray ionization tandem mass spectrometry
(LC-MS/MS) detection of 10 .mu.l of each sample of permethylated
O-glycan species was performed on a Shimadzu Prominence 20 AD LC
and a Thermo GOLD.TM. 3-.mu.m C18 column (2.times.100 mm), coupled
with an ABSCIEX.TM. API-QTRAP.RTM. 5500 tandem mass spectrometer.
The binary method used buffer A (acetonitrile:formic acid: water;
1:0.1:99 (v:v:v)) and buffer B (acetonitrile:formic acid: water;
99:0.1:1 (v:v:v)) with a flow rate at 0.25 mL/min under the
following gradient conditions: O-20 min, 50% to 80% buffer B; 20-28
min, 98% buffer B; 28-39 min, 50% buffer B. The API-QTRAP.RTM. 5500
tandem mass spectrometry conditions were as follows: ion source:
EPI positive mode; curtain gas: 25; source temperature: 600. MRM
transitions for corel T-antigen (as determined by T-antigen
standard) and sialyl-T-antigen (as determined by mass and
fragmentation pattern) (Yoo and Yoon, Bull Korean Chem Soc 26,
1347-1353 (2005)) were: m/z 534/298 and m/z 895/520. Calibration
curves were constructed with 6 concentrations of T-antigen (from
0.0625 to 5 .mu.M). The ST value is based on the ratio of the ST
over the internal standard raffinose peak area, times the raffinose
concentration.
[0162] The permethylated O-glycans were subsequently analyzed by
matrix-assisted laser desorption-ionization (MALDI) time-of-flight
(TOF) mass spectrometry on an Applied Biosystems MALDI-TOF/TOF 4800
Plus (Applied Biosystems, Foster City, Calif.) as described (Xia et
al. Anal Biochem 442, 178-85 (2013)).
Results
[0163] NCAM Immunoblotting:
[0164] Aberrantly sialylated NCAM, detected by immunoblotting of
patients' serum, is the only previously suggested blood-based
marker for GNE myopathy (Valles et al. Genet Test Mol Biomarkers
16, 313-317 (2012)) Immunoblotting of GNE myopathy serum was
performed using the same conditions and NCAM (RNL-1; Santa Cruz
Biotechnology) antibodies as previously employed (Valles et al.,
supra) but similar immunoresponsive bands were not observed (FIG.
7A). This may have been due to different sample handling or
processing, or a different batch of the antibody than that used in
the previous study. However, it was found that a different antibody
to NCAM (H-300; Santa Cruz Biotechnology), detected all three major
(.about.120, 140 and 180 kDa) isoforms of NCAM (Cunningham et al.
Science 236: 799-806 (1987); Small et al. J Cell Biol 105,
2335-2345 (1987)) in human serum samples. Compared to control
serum, all GNE myopathy patient serum samples demonstrated a slight
downshift of the .about.140 kDa NCAM isoform band, similar to a
desialylated (by neuraminidase treatment) control sample (FIG. 4;
FIG. 7B-7C). This downshift likely resulted from different
electrophoretic mobility due to hyposialylation. The .about.120 kDa
and .about.180 kDa isoforms do not appear to be desialylated in GNE
myopathy serum samples.
[0165] Lectin Histochemistry and Lectin Blotting:
[0166] Staining with lectins (i.e., sugar-binding proteins with
ligand specificities for defined carbohydrate sequences (Sharon, J.
Biol. Chem. 282(5), 2753-2764 (2007)) was performed on normal and
GNE myopathy muscle slides to examine the sialylation status. WGA
(wheat germ agglutinin from Triticum vulgaris) predominantly
recognizes terminal sialic acid (Sia) and N-acetylglucosamine
(GlcNAc) on glycans (Sharon, supra; Iskratsch et al., Anal.
Biochem. 386(2), 133-146 (2009); Kronis and Carver, Biochemistry
21(13), 3050-3057 (1982)) SNA (elderberry bark agglutinin from
Sambucus nigra) predominantly recognizes terminal sialic acid (Sia)
in an .alpha.(2,6)-linkage with either galactose (prevalent in
N-linked glycans) or with N-acetylgalactosamine (GalNAc) (found in
O-linked glycans) (Iskratsch et al., Anal. Biochem. 386(2), 133-146
(2009); Kronis et al., Biochemistry 21(13), 3050-3057 (1982);
Shibuya et al., J. Biol. Chem. 262(4), 1596-1601 (1987)). VVA
(hairy vetch agglutinin from Vicia villosa) predominantly binds
GalNAc O-linked to serine or threonine residues of proteins
(Iskratsch et al., op. cit.; Puri et al., FEBS Lett 312(2-3),
208-212 (1992)). Results of control experiments, indicating the
specificity of each lectin, are presented in FIG. 8.
[0167] GNE myopathy muscle, stained with WGA (recognizing most
terminal sialic acids), showed a similar staining pattern as normal
muscle (FIG. 5). However, staining with SNA (binding only
.alpha.(2,6)-linked sialic acid) showed a markedly decreased signal
in patients' muscle slides compared to normal, indicating that only
specific sialylglycans are hyposialylated in GNE myopathy. VVA
staining was almost absent in normal muscle since most glycans are
sialylated, while GNE myopathy muscle showed a significant increase
in staining compared to normal, indicating hyposialylation of
O-linked glycans (FIG. 5).
[0168] Western blots were performed of controls, neuraminidase
treated controls, and GNE myopathy serum proteins, and probed the
blots with WGA, SNA or VVA (FIG. 9). While the neuraminidase
treated control samples showed the expected reduction (for WGA and
SNA) or increase (for VVA) in lectin binding, no significant
differences in lectin binding could be identified in GNE myopathy
patients' serum compared to control serum.
[0169] T/ST Ratios in GNE Myopathy Patients:
[0170] Plasma O-glycan species in control and GNE myopathy patients
were analyzed by LC-MS/MS and MALDI-TOF/TOF. Five O-linked peaks
were observed, at m/z 534, 895, 1256, 1344, and 1706 (FIG. 3A). The
two major peaks in GNE myopathy patients represent the core 1
O-glycan species T-antigen (m/z 534) and the ST-antigen (m/z 895)
(Faid et al., Proteomics 7(11), 1800-1813 (2007); Xia et al., Anal.
Biochem. 442(2), 178-85 (2013)). The relative quantities of T and
monosialylated ST antigens were measured using the LC-MS/MS method
by comparing their intensities to the internal standard raffinose
at m/z 681 (Table 5, Table 6), as well as using purified T-antigen
as external standard to further validate T-antigen quantities.
Except for purified T-antigen, there are no purified standards of
other O-glycan species (e.g., ST-antigen (m/z 895), m/z 1256, 1344
peaks) commercially available at this time. To evaluate the
sialylation of core 1 O-glycan species per patient, the ratio
between T- and monosialylated ST-antigen was obtained. Fifty
control samples (from the normal plasma collection at the Emory
Biochemical Genetics Laboratory) were measured to establish a
normal range for both T-antigen (0.280-1.398 .mu.M), monosialylated
ST antigen (14.145-30.373 .mu.M) and the T/ST ratio (<0.052),
similar ranges as recently previously described (Xia et al.,
supra). An additional 5 control plasma samples from the NM blood
bank were in the normal range for T-antigen, monosialylated
ST-antigen and the T/ST ratio.
[0171] In GNE myopathy plasma, one of the absolute values of either
T- or monsialylated ST-antigen often appeared within the normal
range, but the T/ST ratio was consistently abnormal (>0.052,
note all abnormal samples are >0.06) in all analyzed plasma
samples from untreated patients (FIG. 6B; Table 6; Table 5)
Importantly, the T/ST ratio of one of the untreated GNE myopathy
patients was abnormal (GNE-914a; T/ST=0.100), but shifted to the
normal range 24 hours after intravenous immunoglobulin (IVIG)
therapy on two consecutive days (GNE914b; T/ST=0.0454).
TABLE-US-00008 TABLE 6 Mutations and plasma T and monosialylated ST
values of GNE myopathy patients. ##STR00018## Grey highlight:
Abnormal value .sup.1GNE-914a = plasma value before administration
of IVIG .sup.2GNE-914b = plasma value 24 h after IVIG therapy
[0172] Multiple plasma and also serum samples, including samples
that were collected from the same patients at different time-points
(baseline and 3, 6, and/or 9 months after baseline), were tested
from selected patients. These samples did not show significant
differences in the T/ST ratios (Table 7), indicating that plasma as
well as serum can be used for this assay and that the assay is
reproducible.
TABLE-US-00009 TABLE 7 Plasma and serum T, monosialylated ST and
T/ST ratio values at different time points in selected patients
##STR00019## .sup.1Baseline = timepoint of first blood draw; 3,6,9
months = timepoints of subsequent blood draws after baseline.
.sup.2Serum baseline = T, ST and T/ST ratio values determined in
serum from each patient at the baseline blood draw.
[0173] Major bathers to the diagnosis of GNE myopathy have been the
rarity of the disease and the lack of an inexpensive and
noninvasive diagnostic test. Most GNE myopathy patients escape
diagnosis, with a typical diagnostic delay of approximately 10
years after onset of symptoms (Huizing et al., GNE Myopathy.
Scriver's Online Metabolic and Molecular Bases of Inherited
Disease. ommbid.com (258), (2013)). This leads to anxiety and
unnecessary testing, often involving an invasive muscle biopsy
(Noguchi et al., J. Biol. Chem. 279(12), 402-11407 (2004); Tajima
et al., Am. J. Pathol. 166(4), 1121-1130 (2005); Huizing et al.,
Mol. Genet. Metab. 81(3), 196-202 (2004); Broccolini et al., J.
Neurochem. 105(3), 971-981 (2008); Ricci et al, PLoS One 5(4),
e10055 (2010)). As an alternative, blood-based markers were
explored to aid in diagnosis and monitoring response to
therapy.
[0174] Sialylation on NCAM detected by immunochemistry was
suggested as a muscle--(Broccolini et al., Neurology 75(3), 265-272
(2010)) and blood-based marker for GNE myopathy patients (Valles et
al. Genet. Test. Mol. Biomarkers 16(5), 313-317 (2012)), but
results may vary with the antibodies used, since NCAM has several
membrane bound and soluble tissue-specific isoforms (Cunningham et
al., Science 236(4803): 799-806 (1987); Small et al., J. Cell.
Biol. 105(5), 2335-2345 (1987)). The application of a reported
informative NCAM antibody (RNL-1, (Valles et al., op. cit.) in GNE
myopathy serum samples did not show reproducible data (FIG. 7A-7C),
possibly related to differences in sample processing or antibody
batch. However, the tests with another NCAM antibody (H-300) showed
reactivity for all three major NCAM isoforms in human serum
samples. GNE myopathy patients' sera showed a slight down-shift of
the 140 kDa NCAM isoform, indicating a possible difference of
sialylation on NCAM, resulting in different gel mobility in GNE
myopathy patients (FIG. 4). Interestingly, a downshift of the
.about.140 kDa NCAM isoform was previously reported in muscle
extracts of GNE myopathy patients (Ricci et al. Neurology 66(5),
755-758 (2006); Broccolini et al., op. cit.) indicating a possible
link of this isoform to the disease. The .about.120 kDa and
.about.180 kDa isoforms of serum NCAM appeared not informative for
diagnosis of GNE myopathy. Optimizing specificity and sensitivity
of the immunoreactive .about.140 kDa NCAM band in human serum could
be informative for GNE myopathy.
[0175] Predominantly hyposialylated O-linked glycans are present in
GNE myopathy (Tajima et al., Am. J. Pathol. 166(4), 1121-1130
(2005); Huizing et al., Mol. Genet. Metab. 81(3), 196-202 (2004;
Nemunaitis et al. Hum. Gene Ther. 22(11), 1331-1341 (2011);
Niethamer et al., Mol. Genet. Metab. 107(4), 748-755 (2012)). In
the studies disclosed herein, analysis of O-linked glycan
structures in the plasma were analyzed by a recently developed
semi-quantitative method that determines the ratio of the T- and
ST-antigens (T/ST) (Xia et al., Anal. Biochem. 442(2), 178-85
(2013)). Using this method, mild undersialylation of plasma
O-linked glycan species was demonstrated in all tested GNE myopathy
patients, resulting in abnormally high T/ST ratios (>0.052;
Table 6). Determining the T/ST ratios in GNE myopathy proved robust
and superior to solely semi-quantifying and comparing only the
individual T- and ST-antigen values; while individual T- and
ST-antigen values can be in the normal range in some GNE myopathy
patients (Table 6), the T/ST ratio was abnormal (>0.052) in all
untreated patients. Serum samples from selected GNE myopathy
patients showed similar T/ST ratios (results not shown) to the
corresponding plasma samples, indicating that either serum or
plasma can be used for this assay.
[0176] The fact that some GNE myopathy patients have normal values
of T- or ST-antigen indicates that their undersialylation of
O-linked glycan species is likely mild. It is possible that due to
defects in GNE enzyme activities (Noguchi et al., J. Biol. Chem.
279(12), 11402-11407 (2004); Sparks et al., Glycobiology 15(11),
1102-1110 (2005)), a gradual defect in de novo sialic acid
production occurs in GNE myopathy patients. Some glycans may be
preferentially (under)sialylated, perhaps based on
(tissue-specific) substrate affinity, protein-specific transport
pathways through the Golgi-complex for sialylation, expression of
certain sialyltransferases or neuraminidases, or other mechanisms
(Harduin-Lepers et al., PLoS One 7(8), e44193 (2012); Giacopuzzi et
al., PLoS One 7(8), e44193 (2012); Pshezhetsky et al., Biochemistry
(Mosc) 78(7), 736-745 (2013)). The gradual shortage of tissue-,
protein, or sialyl linkage-specific sialylation of predominantly
O-linked glycans may play a role in the adult onset and muscle
specific symptoms of GNE myopathy. Proteins with significant
O-linked glycosylation may largely be affected and contribute to
the phenotype. In this cohort of GNE myopathy patients, there was
no direct correlation of T/ST plasma ratios to severity and onset
of the disease, nor to GNE gene mutations (Table 6).
[0177] Unfortunately, it is difficult to identify GNE myopathy
patients before the onset of symptoms, but the evaluation of T/ST
ratios in such non-symptomatic patients may indicate the usefulness
of T/ST ratios as an early diagnostic tool for the disease.
[0178] The presence of T-antigen, Tn-antigen and STn-antigens was
utilized as markers for certain cancers. Absolute T-, ST-, Tn-, and
STn-antigen values are significantly altered in different forms or
stages of cancers (Springer et al., J. Mol. Med. (Berl) 75(8),
594-602 (1997); Cao et al., Cancer 76(10), 1700-1708 (1995); Goletz
et al., Adv. Exp. Med. Biol. 535, 147-162 (2003); Imai et al.,
Anticancer Res. 21(2B), 1327-1334 (2001)) but their ratios
(including T/ST) are rarely used in cancer research. T/ST ratios
were informative in patients with classic galactosemia
(galactose-1-phosphate uridylyltransferase (GALT)-deficiency ((Liu
et al. Mol Genet Metab 106, 442-454 (2012)).
[0179] Most such glycosylation disorders present with severe
congenital clinical phenotypes, much different from adult onset GNE
myopathy. Early clinical symptoms of GNE myopathy (waddling gait,
foot drop) are non-specific features of various
neurological/muscular disorders and contribute to the delayed
diagnosis of patients. Such early symptoms in combination with
abnormal plasma T/ST ratios can be indicators for GNE mutation
testing, which will ultimately confirm the diagnosis of GNE
myopathy.
[0180] Sialylation-increasing therapies could normalize the plasma
T/ST ratios in GNE myopathy patients, and possibly indicate
response to therapy. Currently, no therapies are currently approved
for GNE myopathy. Plasma samples were acquired from one GNE
myopathy patient who was part of a previously conducted pilot
clinical trial of intravenous supplementation of sialylated
compounds in the form of immune globulins (WIG;
(http://clinicaltrials.gov/identifier: NCT00195637) (Sparks et al.,
BMC Neurol. 7, 3 (2007)). The sialic acid residues on IgG (.about.8
.mu.mol of sialic acid/g) could presumably be recycled to sialylate
other glycans. While this study showed improvement in strength of
different muscle groups and notable subjective improvement reported
by the patients, no biochemically relevant evidence of
re-sialylation was detected. Plasma from the patient before therapy
had an abnormal T/ST value (0.100), while a plasma sample acquired
24 h after 1 g/kg WIG loading on two consecutive days showed a
normalized T/ST ratio (0.045). Human WIG is N-glycosylated and does
not contain O-linked glycans. Therefore the potential presence of
residual, non-degraded IgG in the patient's plasma did not directly
contribute to the ST-value after therapy. The increased ST values
and decreased T/ST ratios after therapy suggest that sialic acids
on the loaded IgG were processed/recycled to create sialylation of
T-antigens on other glycans. Thus, plasma T/ST ratios could be used
for response to therapy in GNE myopathy patients.
[0181] Other substrate replacement therapies for GNE myopathy
patients are currently in exploratory stages, and include oral
supplementation of sialic acid itself (see the clinicaltrials.gov
website) identifiers: NCT01634750, NCT01236898, and NCT01517880)
and oral supplementation of the sialic acid precursor
N-acetylmannosamine (ManNAc) (see the clinicaltrials.gov website,
identifier: NCT01634750). The T/ST ratios can be used for gauging
response to these therapies.
[0182] Thus, it was demonstrated that the ratio of the
Thomsen-Friedenreich (T)-antigen to its sialylated form,
ST-antigen, detected by mass spectrometry, for example mass
semi-quantitative LC-MS/MS and MALDI-TOF/TOF, is a robust
blood-based (serum or plasma) biomarker informative for diagnosis
and for response to therapy for GNE myopathy. In addition, the
specific hyposialylation of core 1 O-linked glycan species can aid
in elucidating the pathology and adult onset clinical symptoms of
GNE myopathy.
[0183] In view of the many possible embodiments to which the
principles of our invention may be applied, it should be recognized
that illustrated embodiments are only examples of the invention and
should not be considered a limitation on the scope of the
invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
* * * * *
References