U.S. patent application number 16/881472 was filed with the patent office on 2020-09-10 for method for the diagnosis of metachromatic leukodystrophy.
The applicant listed for this patent is CENTOGENE AG. Invention is credited to Hermann Mascher, Arndt Rolfs.
Application Number | 20200284806 16/881472 |
Document ID | / |
Family ID | 1000004853558 |
Filed Date | 2020-09-10 |
![](/patent/app/20200284806/US20200284806A1-20200910-C00001.png)
![](/patent/app/20200284806/US20200284806A1-20200910-C00002.png)
![](/patent/app/20200284806/US20200284806A1-20200910-D00001.png)
![](/patent/app/20200284806/US20200284806A1-20200910-D00002.png)
![](/patent/app/20200284806/US20200284806A1-20200910-D00003.png)
![](/patent/app/20200284806/US20200284806A1-20200910-D00004.png)
![](/patent/app/20200284806/US20200284806A1-20200910-D00005.png)
![](/patent/app/20200284806/US20200284806A1-20200910-D00006.png)
![](/patent/app/20200284806/US20200284806A1-20200910-D00007.png)
![](/patent/app/20200284806/US20200284806A1-20200910-D00008.png)
![](/patent/app/20200284806/US20200284806A1-20200910-M00001.png)
View All Diagrams
United States Patent
Application |
20200284806 |
Kind Code |
A1 |
Rolfs; Arndt ; et
al. |
September 10, 2020 |
Method for the Diagnosis of Metachromatic Leukodystrophy
Abstract
The present invention is related to a method for diagnosing
metachromatic leukodystrophy in a subject comprising a step a),
wherein the step a) comprises detecting a biomarker in a sample
from the subject, wherein the sample is selected from the group
consisting of blood, dried blood, serum and plasma and wherein the
biomarker is different from an enzyme.
Inventors: |
Rolfs; Arndt; (Berlin,
DE) ; Mascher; Hermann; (Traiskirchen, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CENTOGENE AG |
ROSTOCK |
|
DE |
|
|
Family ID: |
1000004853558 |
Appl. No.: |
16/881472 |
Filed: |
May 22, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14651450 |
Jun 11, 2015 |
10690682 |
|
|
PCT/EP2013/003750 |
Dec 11, 2013 |
|
|
|
16881472 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6893 20130101;
G01N 2800/04 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 11, 2012 |
EP |
12 008 257.3 |
Dec 12, 2012 |
EP |
12 008 293.8 |
Mar 21, 2013 |
EP |
13 001 454.1 |
Claims
1. A method for diagnosing metachromatic leukodystrophy in a
subject comprising a step a), wherein the step a) comprises
detecting a biomarker in a sample from the subject, wherein the
sample is selected from the group consisting of blood, dried blood,
serum and plasma and wherein the biomarker is different from an
enzyme.
2. The method according to claim 1, wherein the enzyme is selected
from the group comprising arylsulfatase A,
N-acetyl-alpha-glucosaminidase, arylsulfatase and
beta-glucuronidase.
3. The method according to claim 1, wherein the sample is a serum
sample of the subject, a plasma sample of the subject or a dried
blood sample of the subject.
4. The method according to claim 1, wherein the method comprises a
step b) wherein the step b) comprises determining a level of the
biomarker present in the sample.
5. The method according to claim 1, wherein the level of the
biomarker is indicative whether or not the subject is suffering
from metachromatic leukodystrophy or whether or not the subject is
at risk of suffering from metachromatic leukodystrophy.
6. The method according to claim 1, wherein the biomarker is
detected by means of immunoassay, mass spectrometric analysis,
biochip array, functional nucleic acids and/or a fluorescent
derivative of the biomarker.
7. The method according to claim 6, wherein the biomarker is
detected by means of mass spectrometric analysis, optionally
combined with HPLC.
8. The method according to claim 7, wherein mass spectrometric
analysis is selected from the group comprising SELDI, MALDI,
MALDI-Q TOF, MS/MS, TOF-TOF and ESI-O-TOF.
9. The method according to claim 7, wherein the mass spectrometric
analysis comprises or uses MS/MS.
10. The method according to claim 1, wherein the biomarker is free
lyso-Gb1-sulfatide, wherein lyso-Gb1-sulfatide is of formula (I)
##STR00002##
11. The method according to claim 1, wherein step b) comprises
comparing the level of the biomarker in the sample from the subject
with a cut-off value.
12. The method according to claim 11, wherein the cut-off value for
free lyso-Gb1-sulfatide is 0.05 ng/ml.
13. The method according to claim 1, wherein if the level of the
biomarker in the sample from the subject is higher than the cut-off
value, this is indicative that the subject is suffering from
metachromatic leukodystrophy or is at risk of suffering from
metachromatic leukodystrophy; whereas if the level of the biomarker
in the sample from the subject is lower than the cut-off value,
this is indicative that the subject is not suffering from or is not
at risk of suffering from metachromatic leukodystrophy.
14. The method according to claim 12, wherein the method used in
the detection and/or determination of the level of the biomarker
present in the sample, has a limit of determination for free
lyso-Gb1-sulfatide of 0.05 ng/ml.
15. Use of a biomarker for the diagnosis of metachromatic
leukodystrophy, preferably in a method according to claim 1,
wherein the biomarker is free lyso-Gb1-sulfatide.
16. Use of mass spectrometric analysis for the detection of a
biomarker, wherein the biomarker is free lyso-Gb1-sulfatide and the
mass spectrometric analysis preferably comprises or uses MS/MS.
17. Use according to claim 16, wherein the biomarker is detected in
a sample from a subject, whereby the sample is selected from the
group consisting of a plasma sample from the subject, a serum
sample from the subject and a dried blood sample from the
subject.
18. Use according to claim 16, wherein mass spectrometric analysis
is combined with HPLC.
19. A kit for determining the presence of a biomarker in a sample
from a subject, wherein the kit comprises a) an interaction partner
of the biomarker; b) optionally a solid support comprising at least
one capture reagent attached thereto, wherein the capture reagent
binds the biomarker, and c) instructions for using the solid
support to detect the biomarker, wherein the biomarker is free
lyso-Gb1-sulfatide.
20. The kit according to claim 19, wherein the sample is selected
from the group consisting of a plasma sample from the subject, a
serum sample from the subject and a dried blood sample from the
subject.
Description
[0001] The present invention is related to a method for diagnosing
metachromatic leukodystrophy, a method for determining the
effectiveness of at least one treatment applied to a subject being
positively tested for suffering from or being at risk of suffering
from metachromatic leukodystrophy, a method for determining the
effectiveness of a compound for the treatment of metachromatic
leukodystrophy, the use of mass spectrometric analysis for the
detection of a biomarker, and a kit for determining the presence of
a biomarker in a sample from a subject.
[0002] Lysosomal storage diseases, also referred to herein as
lysosomal storage disorders or LSDs, are a group of rare inherited
metabolic disorders that result from defects in lysosomal function.
LSDs result when a specific organelle in the body's cells--the
lysosome--malfunctions. Some of the more prominent lysosomal
storage diseases are Gaucher's disease and Fabry disease.
[0003] LSDs are caused by lysosomal dysfunction usually as a
consequence of deficiency of a single enzyme required for the
metabolism of lipids, glycoproteins or so-called
mucopolysaccharides. Individually, LSDs occur with frequencies of
about 1:10,000 to 1:250,000, however, as a group the incidence is
about 1:5,000. Most of these disorders are autosomal recessively
inherited; however, a few are X-linked inherited, such as Fabry
disease and Hunter syndrome (MPS II).
[0004] Like other genetic diseases, individuals typically inherit
lysosomal storage diseases from their parents. Although each
disorder results from different gene mutations that translate into
a deficiency in enzyme activity, they all share a common
biochemical characteristic--nearly all lysosomal disorders
originate from an abnormal accumulation of substances inside the
lysosome.
[0005] Lysosomal storage diseases affect mostly children and they
often die at a young and unpredictable age, many within a few
months or years of birth. Many other children die of this disease
following years of suffering from various symptoms of their
particular disorder.
[0006] The symptoms of lysosomal storage disease vary, depending on
the particular disorder and other variables like the age of onset,
and can be mild to severe. They can include developmental delay,
movement disorders, seizures, dementia, deafness and/or blindness.
Some people with lysosomal storage disease have enlarged livers
(hepatomegaly) and enlarged spleens (splenomegaly), pulmonary and
cardiac problems, and bones that develop abnormally.
[0007] There are no causative cures for lysosomal storage diseases
and treatment is mostly symptomatic, although bone marrow
transplantation and enzyme replacement therapy (ERT) have been used
for some indications with good success. In addition, umbilical cord
blood transplantation is being performed at specialized centers for
a number of these diseases. In addition, substrate reduction
therapy (SRT), a method used to decrease the accumulation of
storage material, is currently being evaluated for some of these
diseases. Furthermore, chaperone therapy, a technique used to
stabilize the defective enzymes produced by patients, is being
examined for certain of these disorders. Gene therapy constitutes a
further option for the treatment of these diseases.
[0008] Metachromatic leukodystrophy, also referred to herein as MLD
or Arylsulfatase A deficiency, as used herein, is an LSD which is
caused by a deficiency in lysosomal arylsulfatase A, also referred
to herein as ARSA. ARSA catabolizes sulfatides (Von Figura et al,
"Metachromatic leukodystrophy" In: Scriver C R et al. "The
Metabolic and Molecular Bases of Inherited Disease" 8th edn).
Sulfatides accumulate in multiple tissues including
oligodendrocytes and Schwann cells, provoking demyelination in both
the central and peripheral nervous system (Sedel et al., J Inherit
Metab Dis., 2008 June).
[0009] To date a definitive diagnosis of MLD can only be made by
measurement of ARSA activity on leukocytes together with genetic
confirmation. Since numerous different mutations may be the cause
of a particular lysosomal storage disease the sequencing of the
lysosomal arylsulfatase A gene is applied in MLD in order to
confirm the diagnosis.
[0010] Although there are attempts to apply diagnosis methods based
on associated biochemical abnormalities there is an unmet need for
a simple biochemical test exhibiting highly specific and highly
sensitive detection of said lysosomal storage disease at an early
stage, monitoring progression of the disease and early monitoring
the efficacy of applied therapies.
[0011] Therefore, the identification of biomarkers for the early
detection and diagnosis of MLD holds great promise to improve the
clinical outcome of patients. It is especially important for
patients with vague or no symptoms or to detect patients which fail
to respond to a therapy.
[0012] A biomarker should be technically feasible in many hands,
easy to measure; useful, with a consistent, relative magnitude
between affected and controls, or treated and untreated; reliable,
and accurate clinically, and classifiable as strongly predictive or
prognostic.
[0013] Today, no biomarker for diagnosing MLD is available.
[0014] In Gaucher's disease, another LSD, some lysosomal enzymes,
used as indirect biomarkers, were found to be elevated, including
tartrate-resistant acid phosphatase, hexosaminidase, and a human
chitinase, chitotriosidase. Thus there are attempts to monitor the
reduction of storage cells in tissues by measurement of such
surrogate markers of Gaucher cells like chitotriosidase and CCL18
(C. E. Hollak et al., J. Clin. Invest. 93 (1994) 1288-1292; R. G.
Boot et al., Blood 103 (2004) 33-39). However, beside other
disadvantages in the use of chitotriosidase as a biomarker for
Gaucher's disease, said enzyme accumulates independent of a direct
link to the pathology of Gaucher's disease. Furthermore, up to 35%
of given ethnicities demonstrate a defect of the gene coding for
chitotriosidase resulting in an artificially reduced or
unmeasurable chitotriosidase activity.
[0015] The use of primary storage molecules as biomarker was
assessed for glucosylceramide, in plasma of Gaucher's disease
patients and compared to the level of glucosylceramide in healthy
individuals (Groener et al. Biochim Biophys Acta. 2008
January-February; 1781(1-2):72-8. Epub 2007 Dec. 5.; Plasma
glucosylceramide and ceramide in type 1 Gaucher disease patients:
correlations with disease severity and response to therapeutic
intervention.; Groener J E et al.). Nevertheless, although
glucosylceramide measured in said study was increased in plasma of
said patients, said increase of glucosylceramide was not prominent
and thus the specificity and the sensitivity of the method were low
showing that glucosylceramide is not applicable as a biomarker for
Gaucher's disease.
[0016] Already in 1989 Rosengren et al. (lyso-sulfatide
(galactosylsphingosine-3-O-sulfate) from metachromatic
leukodystrophy and normal human brain, Rosengren B, Fredman P,
Mansson JE, Svennerholm L.; J Neurochem. 1989 April;
52(4):1035-41.) showed that in lipidoses not only the catabolism of
the major sphingolipid but also its lyso-compound is affected.
Nevertheless, said study concluded that the lyso-compounds do not
play a key role in the pathogenetic mechanisms in the
sphingolipidoses. Thus, said lyso-compounds might not be suitable
biomarkers for diagnosis of sphingolipidoses such as Gaucher's
disease.
[0017] It is important to note that until today no use of a highly
specific and highly sensitive biomarker and no method for the
diagnosis of MLD is available beside the methods described above
that exhibit an unsatisfactory limit of detection, sensitivity
and/or specificity and thus proved to be unsuitable for clinical
application.
[0018] Accordingly, there is need for a fast, simple and more
importantly reliable method for the diagnosis of MLD.
[0019] In the light of the above, the problem underlying the
present invention is to provide a method for the diagnosis of
MLD.
[0020] It is a still further problem underlying the present
invention to provide a method which allows to determine whether or
not the subject is suffering from MLD or whether or not the subject
is at risk of suffering from MLD.
[0021] A further problem underlying the present invention is to
provide a method for determining the course and prognosis of
MLD.
[0022] A still further problem underlying the present invention is
to provide a method for determining rather quickly the
effectiveness of at least one treatment applied to a subject being
positively tested for suffering from or being at risk of developing
MLD.
[0023] A further problem underlying the present invention is to
provide a method for determining the effectiveness of a compound
for the treatment of MLD.
[0024] Another problem underlying the present invention is to
provide a biomarker which allows the specific and sensitive
diagnosis of MLD.
[0025] A still further problem underlying the present invention is
a kit which comprises a compound which interacts with a biomarker
which is specific and sensitive for MLD.
[0026] Preferably, the biomarker of each and any problem underlying
the present invention and the above problems in particular, is
different from an enzyme, more preferably the biomarker is
different from an enzyme selected form the group comprising
arylsulfatase A, N-acetyl-alpha-glucosaminidase, arylsulfatase and
beta-glucuronidase.
[0027] These and other problems are solved by the subject matter of
the attached independent claims. Preferred embodiments may be taken
from the attached dependent claims. Further aspects of the
invention and various embodiments thereof are disclosed in the
following. In particular, further aspects and embodiments are
presented in the following and referred to as embodiment followed
by a counter, i.e. "Embodiment 1" to "Embodiment 83", each and any
thereof equally constitutes a solution to these and other
problems.
Embodiment 1
[0028] A method for diagnosing metachromatic leukodystrophy in a
subject comprising [0029] a step a), wherein the step a) comprises
detecting a biomarker in a sample from the subject.
Embodiment 2
[0030] The method according to embodiment 1, wherein the method
comprises [0031] a step b) wherein the step b) comprises
determining a level of the biomarker present in the sample.
Embodiment 3
[0032] The method according to any one of embodiments 1 or 2,
wherein the level of the biomarker is indicative whether or not the
subject is suffering from metachromatic leukodystrophy or whether
or not the subject is at risk of suffering from metachromatic
leukodystrophy.
Embodiment 4
[0033] The method according to any one of embodiments 1 to 3,
wherein the sample from the subject is a sample from a subject who
has previously been treated for metachromatic leukodystrophy or a
sample from a subject who has previously been diagnosed for
metachromatic leukodystrophy.
Embodiment 5
[0034] The method according to any one of embodiments 1 to 3,
wherein the sample from the subject is a sample from a subject who
has not previously been treated for metachromatic leukodystrophy or
a sample from a subject who has not been previously diagnosed for
metachromatic leukodystrophy.
Embodiment 6
[0035] The method according to any one of embodiments 1 to 5,
wherein the method comprises [0036] a step c), wherein the step c)
comprises applying, maintaining, reducing, elevating or not
applying a therapy based on whether the subject is suffering from
metachromatic leukodystrophy or is at risk of suffering from
metachromatic leukodystrophy.
Embodiment 7
[0037] The method according to any one of embodiments 1 to 6,
wherein the method comprises [0038] a step d), wherein the step d)
comprises detecting the biomarker in a sample from the subject
after a therapy has been applied, maintained, reduced, elevated or
not applied in step c).
Embodiment 8
[0039] The method according to any one of embodiments 1 to 7,
wherein the method comprises [0040] a step e), wherein the step e)
comprises determining a level of the biomarker in the sample from
the subject after a therapy has been applied, maintained, reduced,
elevated or not applied in step c).
Embodiment 9
[0041] The method according to embodiment 8, wherein the method
comprises [0042] a step f), wherein the step f) comprises
determining whether the level of the biomarker determined in step
b) is lower than the level of the biomarker determined in step
e).
Embodiment 10
[0043] The method according to embodiment 9, wherein the method
comprises [0044] a step g), wherein the step g) comprises applying,
maintaining, reducing, elevating or not applying a therapy based on
step f).
Embodiment 11
[0045] The method according to any one of embodiments 1 to 10,
wherein the biomarker is detected by means of immunoassay, mass
spectrometric analysis, biochip array, functional nucleic acids
and/or a fluorescent derivative of the biomarker.
Embodiment 12
[0046] The method according to embodiment 11, wherein the biomarker
is detected by means of mass spectrometric analysis.
Embodiment 13
[0047] The method according to embodiment 12, wherein mass
spectrometric analysis is selected from the group comprising SELDI,
MALDI, MALDI-Q TOF, MS/MS, TOF-TOF and ESI-O-TOF.
Embodiment 14
[0048] The method according to embodiment 13, wherein the mass
spectrometric analysis comprises or uses MS/MS.
Embodiment 15
[0049] The method according to any one of embodiments 1 to 14,
wherein the method comprises protein precipitation and/or HPLC.
Embodiment 16
[0050] The method according to any one of embodiments 1 to 15,
wherein the method comprises protein precipitation, HPLC and
MS/MS.
Embodiment 17
[0051] The method according to any one of embodiments 1 to 16,
wherein the subject is a human.
Embodiment 18
[0052] The method according to any one of embodiments 1 to 17,
wherein step d) comprises detecting the biomarker in a sample
comprises subjecting the sample to a protein precipitation step,
precipitating protein from the sample, providing a supernatant of
the sample, subjecting the supernatant of the sample to HPLC and
MS/MS and determining the level of the biomarker that is present in
the supernatant of the sample.
Embodiment 19
[0053] The method according to any one of embodiments 1 to 18,
wherein the biomarker is free lyso-Gb1-sulfatide.
Embodiment 20
[0054] A method for diagnosing metachromatic leukodystrophy in a
subject, wherein the method comprises the following steps: [0055]
i) adding an internal standard to a sample from the subject,
wherein the sample from the subject is selected from the group
comprising plasma, serum and blood; [0056] ii) optionally mixing
the sample containing the internal standard; [0057] iii) subjecting
the sample to a protein precipitation step, whereby protein from
the sample is precipitated and a first supernatant of the sample is
provided; [0058] iv) optionally subjecting the first supernatant of
the sample or at least a part thereof to a first separation step
which provides a second supernatant, whereby preferably the first
separation step is a step of centrifugation; [0059] v) subjecting
the first supernatant and/or the second supernatant, or at least a
part thereof, to a second separation step, wherein the second
separation step comprises injecting at least a part of the first
supernatant and/or at least a part of the second supernatant into
an HPLC-MS/MS system and using an HPLC column with a gradient from
acidic water to acetonitrile/acetone; wherein the HPLC column is
preferably an HPLC column selected from the group comprising a C8
HPLC column and a C18 HPLC column, and wherein the second
separation step provides a separated sample; [0060] vi) subjecting
the separated sample to MS/MS, wherein MS/MS comprises electrospray
ionization and Multiple Reaction Monitoring; and comprising [0061]
a step a), wherein the step a) comprises detecting a biomarker in a
sample from the subject, and optionally [0062] a step b), wherein
the step b) comprises determining a level of the biomarker present
in the sample, wherein the biomarker is free lyso-Gb1-sulfatide,
and wherein the method is preferably a method according to any one
of embodiments 1 to 19;
Embodiment 21
[0063] The method according to any one of embodiment 20, wherein
the internal standard comprises lyso-Gb2.
Embodiment 22
[0064] The method according to any one of embodiments 1 to 21,
wherein step b), step c) and/or step e) comprises comparing the
level of the biomarker in the sample from the subject with a
cut-off value.
Embodiment 23
[0065] The method according to any one of embodiments 1 to 22,
preferably 22, wherein if the level of the biomarker in the sample
from the subject is higher than the cut-off value this is
indicative that the subject is suffering from metachromatic
leukodystrophy or is at risk of suffering from metachromatic
leukodystrophy.
Embodiment 24
[0066] The method according to any one of embodiments 1 to 22,
preferably 22, wherein if the level of the biomarker in the sample
from the subject is lower than the cut-off value this is indicative
that the subject is not suffering from or is not at risk of
suffering from metachromatic leukodystrophy.
Embodiment 25
[0067] The method according to any one of embodiments 1 to 24,
wherein the cut-off value is such that a or the sensitivity for
diagnosing metachromatic leukodystrophy in a subject is preferably
from about 98.5% to 100%, more preferably 99.5% to 100%, and/or
such that a or the specificity for diagnosing metachromatic
leukodystrophy in a subject is from 99.4% to 100%, preferably
100%.
Embodiment 26
[0068] The method according to any one of embodiments 1 to 25,
wherein step b) and/or step c) and/or step e) comprise(s) that a
level of the biomarker in said subject is compared to a level of
the biomarker detected in a sample from a control sample.
Embodiment 27
[0069] The method according to embodiment 26, wherein the control
sample is a sample from a subject not having metachromatic
leukodystrophy.
Embodiment 28
[0070] The method according to embodiment 26, wherein the control
sample is a sample from a subject having metachromatic
leukodystrophy carrier.
Embodiment 29
[0071] The method according to any one of embodiments 26 to 28,
wherein if the level of the biomarker in the sample from the
subject is higher than the level of the biomarker in the control
sample this is indicative that the subject is suffering from and/or
is at risk of suffering from metachromatic leukodystrophy.
Embodiment 30
[0072] The method according to any one of embodiments 1 to 29,
preferably to embodiment 29, wherein the sample from the subject is
selected from the group comprising blood, a blood product, urine,
saliva, cerebrospinal fluid, stool, tissue sample and lymph.
Embodiment 31
[0073] The method according to embodiment 30, wherein the sample
from the sample from the subject is selected from the group
comprising blood and a blood product.
Embodiment 32
[0074] The method according to any one of embodiments 30 to 31,
wherein the blood product is selected from the group comprising
plasma, serum and dried blood.
Embodiment 33
[0075] The method according to any one of embodiments 1 to 32,
preferably 32, wherein the method has a limit of determination for
free lyso-Gb1-sulfatide of 0.05 ng/ml.
Embodiment 34
[0076] The method according to any one of embodiments 1 to 33,
wherein the method is for the diagnosis of metachromatic
leukodystrophy carrier and wherein the biomarker is free
lyso-Gb1-sulfatide and the cut-off value is 0.05 ng/ml, and wherein
the sample from the subject is preferably serum or plasma.
Embodiment 35
[0077] The method according to any one of embodiments 30 to 31,
wherein the blood is whole blood, plasma, serum or dried blood,
preferably plasma or serum.
Embodiment 36
[0078] The method according to embodiment 35, wherein the whole
blood, plasma or serum is collected on a dry blood filter card.
Embodiment 37
[0079] A method for determining the course of metachromatic
leukodystrophy in a subject, wherein the method comprises [0080] a
step a), wherein the step a) comprises determining at several
points in time a level of a biomarker present in a sample from the
subject.
Embodiment 38
[0081] The method according to embodiment 37, wherein the subject
has been previously treated for metachromatic leukodystrophy and/or
wherein the subject has been previously diagnosed for metachromatic
leukodystrophy.
Embodiment 39
[0082] The method according to embodiment 37, wherein the subject
has not been previously treated for metachromatic leukodystrophy
and/or wherein the subject has not been previously diagnosed for
metachromatic leukodystrophy.
Embodiment 40
[0083] The method according to any one of embodiments 37 to 39,
wherein the method comprises [0084] a step b), wherein the step b)
comprises applying, maintaining, reducing, elevating or not
applying a therapy based on whether the subject is suffering from
metachromatic leukodystrophy or is at risk of suffering from
metachromatic leukodystrophy.
Embodiment 41
[0085] The method according to any one of embodiments 37 to 40,
wherein the method comprises [0086] a step c), wherein the step c)
comprises detecting the biomarker in a sample from the subject
after a therapy has been applied, maintained, reduced, elevated or
not applied in step b).
Embodiment 42
[0087] The method according to any one of embodiments 37 to 41,
wherein the method comprises [0088] a step d), wherein the step d)
comprises determining a level of the biomarker in the sample from
the subject after a therapy has been applied, maintained, reduced,
elevated or not applied in step b).
Embodiment 43
[0089] The method according to any one of embodiments 37 to 42,
wherein the method comprises [0090] a step e), wherein the step e)
comprises determining whether the level of the biomarker determined
in step a) is lower than the level of the biomarker determined in
step d).
Embodiment 44
[0091] The method according to any embodiment 43, wherein the
method comprises [0092] a step f), wherein the step f) comprises
applying, maintaining, reducing, elevating or not applying a
therapy based on step e).
Embodiment 45
[0093] The method according to any one of embodiments 37 to 44,
wherein the biomarker is free lyso-Gb1-sulfatide.
Embodiment 46
[0094] The method according to any one of embodiments 37 to 45,
wherein the method comprises determining the level of free
lyso-Gb1-sulfatide.
Embodiment 47
[0095] The method according to any one of embodiments 37 to 46,
wherein the method comprises detecting free lyso-Gb1-sulfatide in
the sample from the subject.
Embodiment 48
[0096] The method according to any one of embodiments 37 to 47,
wherein the biomarker is detected by means of immunoassay, mass
spectrometric analysis, biochip array, functional nucleic acids
and/or a fluorescent derivative of the biomarker.
Embodiment 49
[0097] The method according to embodiment 48, wherein the biomarker
is detected by means of mass spectrometric analysis.
Embodiment 50
[0098] The method according to embodiment 49, wherein mass
spectrometric analysis is selected from the group consisting of
SELDI, MALDI, MALDI-Q TOF, MS/MS, TOF-TOF and ESI-O-TOF.
Embodiment 51
[0099] The method according to embodiment 50, wherein the mass
spectrometric analysis comprises or uses MS/MS.
Embodiment 52
[0100] The method according to any one of embodiments 37 to 51,
wherein the method comprises protein precipitation and/or HPLC.
Embodiment 53
[0101] The method according to any one of embodiments 37 to 52,
wherein the method comprises protein precipitation, HPLC and
MS/MS.
Embodiment 54
[0102] The method according to any one of embodiments 37 to 53,
wherein the subject is a human.
Embodiment 55
[0103] The method according to any one of embodiments 37 to 54,
wherein step d) comprises detecting the biomarker in a sample
comprises subjecting the sample to a protein precipitation step,
precipitating protein from the sample, providing a supernatant of
the sample, subjecting the supernatant of the sample to HPLC and
MS/MS and determining the level of the biomarker that is present in
the supernatant of the sample.
Embodiment 56
[0104] A method for determining the effectiveness of at least one
treatment applied to a subject being positively tested for
suffering from or being at risk of suffering from metachromatic
leukodystrophy comprising a step a), wherein the step a) comprises
detecting at several points in time a level of a biomarker present
in a sample from the subject.
Embodiment 57
[0105] The method according to embodiment 56, wherein the method
comprises [0106] a step b), wherein the step b) comprises
determining at several points in time a level of a biomarker
present in a sample from the subject.
Embodiment 58
[0107] The method according to any one of embodiments 56 or 57,
wherein the biomarker is free lyso-Gb1-sulfatide.
Embodiment 59
[0108] The method according to any one of embodiments 56 to 58,
wherein the subject has been previously treated for metachromatic
leukodystrophy or diagnosed for metachromatic leukodystrophy.
Embodiment 60
[0109] The method according to any one of embodiments 56 to 58,
wherein the subject has not been previously treated for
metachromatic leukodystrophy or wherein the subject has not been
previously diagnosed for metachromatic leukodystrophy.
Embodiment 61
[0110] The method according to any one of embodiments 56 to 60,
wherein the method comprises [0111] a step d), wherein the step d)
comprises applying, maintaining, reducing, elevating or not
applying at least one treatment applied to the subject based on the
decrease in the level of the biomarker as determined in step
b).
Embodiment 62
[0112] The method according to any one of embodiments 56 to 61,
wherein the method comprises [0113] a step e), wherein the step e)
comprises detecting the biomarker in the sample from the subject,
wherein the sample has been taken prior to the beginning of the
treatment after applying, maintaining, reducing, elevating or not
applying at least one treatment in step d) and, optionally
determining a level of a biomarker present in a sample from the
subject.
Embodiment 63
[0114] The method according to any one of embodiments 56 to 62,
wherein the treatment is selected from the group comprising enzyme
replacement therapy, substrate reduction therapy, chaperone
therapy, gene therapy, stem cell transplantation of DNA/RNA
skipping.
Embodiment 64
[0115] The method according to any one of embodiments 56 to 63,
wherein the method comprises [0116] a step f), wherein the step 0
comprises determining whether the level of the biomarker determined
in step b) is lower than the level of the biomarker determined in
step e).
Embodiment 65
[0117] The method according to embodiment 64, wherein the method
comprises [0118] a step g) wherein step g) comprises applying,
maintaining, reducing, elevating or not applying at least one
treatment applied to the subject based on step f).
Embodiment 66
[0119] The method according to any one of embodiments 56 to 65,
wherein the biomarker is detected by means of immunoassay, mass
spectrometric analysis, biochip array, functional nucleic acids
and/or a fluorescent derivative of the biomarker.
Embodiment 67
[0120] The method according to embodiment 66, wherein the biomarker
is detected by means of mass spectrometric analysis.
Embodiment 68
[0121] The method according to embodiment 67, wherein mass
spectrometric analysis is selected from the group consisting of
SELDI, MALDI, MALDI-Q TOF, MS/MS, TOF-TOF and ESI-O-TOF.
Embodiment 69
[0122] The method according to embodiment 68, wherein the mass
spectrometric analysis comprises or uses MS/MS.
Embodiment 70
[0123] The method according to any one of embodiments 56 to 69,
wherein the method comprises protein precipitation and/or HPLC.
Embodiment 71
[0124] The method according to any one of embodiments 56 to 70,
wherein the method comprises protein precipitation, HPLC and
MS/MS.
Embodiment 72
[0125] The method according to any one of embodiments 56 to 71,
wherein the subject is a human.
Embodiment 73
[0126] The method according to any one of embodiments 56 to 72,
wherein the step of detecting the biomarker in the sample from the
subject comprises precipitating protein from the sample from the
subject, wherein precipitating protein from the sample provides a
supernatant of the sample; subjecting a volume of the supernatant
to HPLC and MS/MS and determining the level of the biomarker that
is present in the sample from the subject.
Embodiment 74
[0127] A method of determining the effectiveness of a compound for
the treatment of metachromatic leukodystrophy, wherein the method
comprises the following steps: [0128] a) determining a level of a
biomarker in a sample form a subject having metachromatic
leukodystrophy; [0129] b) administering to said subject said
compound; [0130] c) determining again the level of the biomarker in
a sample from the subject after the compound has been administered
to the subject; and [0131] d) determining whether the level of the
biomarker determined in step c) is lower than the level of the
biomarker determined in step a); wherein if a level of the
biomarker determined in step c) is lower than the level of the
biomarker determined in step a) this indicates the effectiveness of
said compound.
Embodiment 75
[0132] The method according to embodiment 74, wherein the biomarker
is free lyso-Gb1-sulfatide.
Embodiment 76
[0133] The method according to any one of embodiments 74 to 75,
wherein the method comprises determining a level of the biomarker
in a control sample.
Embodiment 77
[0134] The method according to any one of embodiments 1 to 76,
wherein the biomarker is free lyso-Gb1-sulfatide, wherein if the
level of the biomarker in the sample from the subject is higher
than the cut-off value of 0.05 ng/ml this is indicative that the
subject is suffering from metachromatic leukodystrophy.
Embodiment 78
[0135] Use of mass spectrometric analysis for the detection of a
biomarker, wherein the biomarker is free lyso-Gb1-sulfatide.
Embodiment 79
[0136] Use according to embodiment 78, wherein the detection
comprises the use of HPLC.
Embodiment 80
[0137] Use according to any one of embodiments 78 to 79, wherein
the mass spectrometric analysis comprises or uses MS/MS.
Embodiment 81
[0138] Use of a biomarker for the diagnosis of metachromatic
leukodystrophy, preferably in a method according to any one of
embodiments 1 to 77, wherein the biomarker is free
lyso-Gb1-sulfatide.
Embodiment 82
[0139] A kit for determining the presence of a biomarker in a
sample from a subject, wherein the kit comprises [0140] a) an
interaction partner of the biomarker; [0141] b) optionally a solid
support comprising at least one capture reagent attached thereto,
wherein the capture reagent binds the biomarker; and [0142] c)
instructions for using the solid support to detect the biomarker,
[0143] wherein the biomarker is free lyso-Gb1-sulfatide.
Embodiment 83
[0144] The kit according to embodiment 82, wherein the kit is for
[0145] a) use in a method for diagnosing metachromatic
leukodystrophy; [0146] b) use in a method for determining the
course of metachromatic leukodystrophy in a subject; and/or [0147]
c) use in a method for determining the effectiveness of at least
one treatment applied to a subject, wherein preferably the method
of a), b) and/or c) is a method according to any one of embodiments
1 to 77.
[0148] It is within the present invention that the biomarker as
used or referred to in connection with each and any aspect of the
invention and each and any embodiment of the invention is
preferably different from an enzyme, more preferably the biomarker
is different from an enzyme selected form the group comprising
arylsulfatase A, N-acetyl-alpha-glucosaminidase, arylsulfatase and
beta-glucuronidase.
[0149] Preferably, the subject of each and any aspect of the
invention and each and any embodiment of the invention is one
suffering from a mutation selected form the group comprising the
following MLD mutations [0150] c.465+1G>A (5) [0151] p.G309S (4)
[0152] p.T393G (3) [0153] p.L113P (2) [0154] p.Q159X (2) [0155]
p.D257E (2) [0156] p.K304R (2) [0157] p.G311S (2) [0158] p.A316D
(2) [0159] p.Y381fs (2) [0160] p.F387fs (2) [0161] p.H397Y (2)
[0162] p.F400L (2) [0163] p.P426L (2) [0164] c.979+1G>A (2)
[0165] p.P84L (1) [0166] p.S98F (1) [0167] p.G1245 (1) [0168]
p.G129R (1) [0169] p.C156Y (1) [0170] p.C158X (1) [0171] p.W195C
(1) [0172] p.R246H (1) [0173] p.T276M (1) [0174] p.T306M (1) [0175]
p.G309D (1) [0176] p.E314D (1)
[0177] These and other mutations are, for example, described in
Pediatric Neurology, Part III: Handbook of Clinical Neurology, Eds.
Olivier Dulac Maryse Lassonde Harvey B. Sarnat; Elsevier, 2013
[0178] The likelihood that the specific mutation is shown by an MLD
patient is indicated in brackets in the above list.
[0179] The present inventors have surprisingly found that free
lyso-Gb1-sulfatide, also referred to herein preferably as free
lyso-glycocerebroside-sulfatide, constitutes a biomarker which
allows for a method for diagnosing MLD in a subject, more
specifically diagnosing MLD in a subject with high specificity and
sensitivity using said free lyso-Gb1-sulfatide as the biomarker.
Reliance on free lyso-Gb1-sulfatide as a biomarker in the diagnosis
of MLD is superior over any method for the diagnosis of MLD based
on an enzyme and arysulfatase A in particular. Such superiority of
a diagnosis based on free lyso-Gb1-sulfatide is shown in terms of
both sensitivity and specificity. One of the reasons for the
observed superiority of a free lyso-Gb1-sulfatide based diagnosis
over an arylsulfatase A based diagnosis of MLD is the existence
arylsulfatase A pseudodeficiencies, where a decrease in
arylsulfatase A activity is--incorrectly--taken as an indication of
MLD. Similar short-comings are observed in case of MLD diagnosis
based on genetics: Some of the observed mutations of arylsulfatase
A result in non-pathogenic pseudodeficiencies of arylsulfatase
A.
[0180] The present inventors have also surprisingly found that free
lyso-Gb1-sulfatide, which can be detected by the methods of the
present invention, is circulating in the blood, and plasma and
serum in particular, of a subject in a concentration of
approximately 1/1000 of total Gb1-sulfatide. Moreover, the present
inventors have surprisingly found that, unlike total Gb1-sulfatide,
free lyso-Gb1-sulfatide which is present in the blood, and plasma
and serum in particular, of a subject is useful in a method for
diagnosing MLD in a subject comprising a step of detecting a
biomarker in a sample from the subject, wherein the biomarker is
free lyso-Gb1-sulfatide. The present inventors have also
surprisingly found that the level of free lyso-Gb1-sulfatide
determined in the sample from a subject by the methods of the
present invention allows for diagnosing MLD with high sensitivity
and high specificity.
[0181] In so far the present invention turns away from the teaching
of the prior art in that the method of the present invention
comprises determining the level of a lyso-compound and using said
lyso-compound as a biomarker for diagnosis of a LSD. More
specifically, the present inventors have surprisingly found that
determining the level of free lyso-Gb1-sulfatide in a sample from a
subject allows for diagnosing MLD with high sensitivity and high
specificity.
[0182] It is also the merit of the present inventors of having
recognized that a fraction of total Gb1-sulfatide which is
accumulated in MLD, is present as a molecule in a free lyso form
thereof, i.e. free lyso-Gb1-sulfatide, and is circulating in the
blood, and plasma and serum in particular, of a subject in said
free lyso form besides Gb1-sulfatide.
[0183] The term "lysosomal storage disorder", also referred to
herein as "lysosomal storage disease" or "LSD", as preferably used
herein, refers to genetic diseases and metabolic disorders that
result from defects in lysosomal function. Lysosomal storage
disorders are caused by lysosomal dysfunction usually as a
consequence of deficiency of a single enzyme required for the
metabolism of lipids, glycoproteins or so-called
mucopolysaccharides. Like other genetic diseases, individuals
inherit lysosomal storage diseases from their parents. Although
each disorder results from different gene mutations that translate
into a deficiency in enzyme activity, they all share a common
biochemical characteristic--all lysosomal disorders originate from
an abnormal accumulation of substances inside the lysosome.
[0184] MLD is an autosomal recessively inherited LSD which is
commonly listed in the family of leukoencephalopathies. The term
leukoencephalopathy means a disease or disorder that selectively or
predominantly involves the white matter of the brain. It is
associated with a group of diseases that affect the myelin itself,
oligodendrocytes, astrocytes or even axons. The main acquired
causes of leukoencephalopathies include inflammatory diseases,
vascular diseases, infections, neoplasias and toxic causes
(reviewed in Filley and Kleinschmidt-DeMasters, 2001, N Engl J
Med). Hereditary leukoencephalopathies can be separated into three
categories (Baumann and Turpin, 2000, J Neurol; Schiffmann and van
der Knaap, 2004, Curr Opin Neurol; Sedel et al, 2005, Rev Neurol):
(1) leukoencephalopathies characterized clinically, radiologically
or pathologically but for which the gene causing the
leukoencephalopathy is still unknown; (2) leukoencephalopathies
caused by genes coding for proteins not directly involved in
metabolic pathways and for which the diagnosis relies directly on
gene analysis; and (3) leukoencephalopathies caused by genes coding
for enzymes or proteins involved in the cell metabolism and for
which the diagnosis relies mostly on biochemical analysis of plasma
and urines samples. The third category corresponds to inborn errors
of metabolism, also referred to herein as IEMs, which are important
to recognize because specific treatments often exist (Sedel et al.,
2007, Nat Clin Pract Neurol). Most IEMs causing
leukoencephalopathies begin in childhood and have been described by
neuropaediatricians. However, late-onset forms also exist that
display different clinical and radiological features, sometimes
very far from the classical paediatric description. With the
exception of some leukoencephalopathies caused by certain lysosomal
or peroxisomal disorders, neurologists are usually poorly familiar
with IEMs.
[0185] MLD belongs to the third group of leukoencephalopathies,
i.e. leukoencephalopathies caused by genes coding for enzymes or
proteins involved in the cell metabolism. MLD is caused by a
deficiency in lysosomal arylsulfatase A, also referred to herein as
ARSA. ARSA catabolizes sulfatides (Von Figura et al, 2001,
supra).
[0186] The incidence of the disease is around 1/100 000 and adult
forms represent about 20% of cases. Clinical onset can be as late
as the seventh decade of life (Bosch and Hart, 1978, Arch Neurol;
Von Figura et al, 2001, supra). In adults, first symptoms are
usually psychiatric, mimicking schizophrenia with delusion,
hallucinations, disorganized behaviour and social dysfunction
(Baumann et al 1991, Dev Neurosci). The clinical picture is
completed after several years or decades by cognitive deficits as
well as motor signs such as spastic paraparesis, cerebellar ataxia
or mild demyelinating polyneuropathy. Motor onset forms of the
disease are preferentially associated with the homozygous mutation
P426L, whereas psychiatric forms are linked to the I179S mutation
(Rauschka et al, 2006, Neurology). Magnetic Resonance Imaging shows
a bilateral periventricular leukoencephalopathy with frontal
predominance and cerebral atrophy. Importantly U-fibres are
relatively spared at least at early stages of the disease (Sedel et
al, 2001, supra).
[0187] To date, the only treatment which can be proposed is bone
marrow transplantation, with few successes obtained in late-onset
forms of the disease (Kidd et al., 1998, Arch Neurol).
[0188] Diagnosis of MLD according to the prior art is based on the
measurement of ARSA activity on leukocytes. However, about 15% of
people in Europe and the United States display low ARSA activity
without clinical symptoms and no tissue or urine accumulation of
sulfatides (Von Figura et al., 2001, supra). These
pseudodeficiencies are caused by certain polymorphism within the
ARSA coding gene (Von Figura et al., 2001, supra).
[0189] Thus the diagnosis of MLD in a patient with low ARSA
activity according to the prior art requires the demonstration of
high urinary excretion of sulfatides or molecular analysis of the
ARSA gene.
[0190] Deficiency in saposin B, an activator necessary to activate
sulfatides degradation, can cause MLD despite normal ARSA activity
(Deconinck et al., 2007, Eur J Paediatr Neurol). Although such
deficiency has not been described in adults to our knowledge, it
should be suspected in patients with leukodystrophy and high
urinary excretion of sulfatides.
[0191] Like many other genetic disorders that affect lipid
metabolism, there are several forms of MLD, which are late
infantile, juvenile, and adult.
[0192] In the late infantile form, which is the most common form of
MLD (50-60%), affected children begin having difficulty walking
after the first year of life, usually at 15-24 months. Symptoms
include muscle wasting and weakness, muscle rigidity, developmental
delays, progressive loss of vision leading to blindness,
convulsions, impaired swallowing, paralysis, and dementia. Children
may become comatose. Untreated, most children with this form of MLD
die by age 5, often much sooner.
[0193] Children with the juvenile form of MLD (onset between 3 and
10 years of age) usually begin with impaired school performance,
mental deterioration, and dementia and then develop symptoms
similar to the late infantile form but with slower progression. Age
of death is variable, but normally within 10 to 15 years of symptom
onset although some juveniles can live for several decades or
longer after onset.
[0194] The adult form commonly begins after age 16 as a psychiatric
disorder or progressive dementia. Adult-onset MLD progresses more
slowly than the late infantile and juvenile forms, with a
protracted course of a decade or more.
[0195] Palliative care can help with many of the symptoms and
usually improves quality and longevity of life.
[0196] That MLD is inherited in an autosomal recessive pattern
means that both copies or both alleles of the gene must be mutated
or altered in such a way that function is impaired, in contrast to
a polymorphism, in which the nucleotide sequence is altered but
causes no functional disruption, for a person to be affected by the
disorder. Most often, the parents of a child with an autosomal
recessive disorder are not affected but are carriers of one copy of
the altered gene. Such carrier is referred to herein as MLD
carrier. If both parents are carriers, there is a 25% chance with
each pregnancy for an affected child. Genetic counseling and
genetic testing is recommended for families who may be carriers of
MLD.
[0197] Sulfatides accumulate in multiple tissues including
oligodendrocytes and Schwann cells, provoking demyelination in both
the central and peripheral nervous system (Sedel et al., supra).
Sulfatides are a class of sulfated galactosylceramides synthesized
primarily in the oligodendrocytes in the central nervous system.
Sulfatides are a type of sulfolipid.
[0198] Gb1-sulfatide consists of a ceramide core, i.e. sphingosine
bound to a fatty acid via an amide linkage and one glycosyl residue
at the 1-hydroxyl moiety. A sulfate moiety is present at the
C.sub.3-atom of the glycosyl moiety. Preferably, the glycosyl
moiety is either a galactosyl moiety or a glucosyl moiety. More
preferably, the glycosyl moiety is a galactosyl moiety.
[0199] It will be understood by a person skilled in the art that
the term "lyso-Gb1-sulfatide" as used herein, preferably in
connection with the various methods of the invention, preferably
means that the molecule is present in its free amino form. More
precisely, lyso-Gb1-sulfatide as used herein, preferably differs
from Gb1-sulfatide in that no fatty acid moiety is linked to
the--primary--amino group of the sphingosine moiety of the
molecule. Furthermore, lyso-Gb1-sulfatide is also referred to
herein as glycosylsphingosine-sulfatide. In a preferred embodiment
of the present invention, lyso-Gb1 sulfatide is of formula (I)
##STR00001##
and is also referred to as lyso-Galactosyl-ceramide or
lyso-galactosyl ceramide sulfatide or galactosyl sphingosine
sulfatide.
[0200] It will be acknowledged by a person skilled in the art that
depending on the analytical method used, it might not be possible
to make a distinction whether a detected
glycosylsphingosine-sulfatide contains a galactosyl moiety or a
glucose moiety as the glycosyl moiety. Such a method is, for
example, HPLC-MS/MS. Because of this, the term lyso-Gb1 sulfatide
encompasses in its more general meaning as used herein
galactosylsphingosine-sulfatide and/or
glucosylsphingosine-sulfatide, whereby preferably the term lyso-Gb1
sulfatide means a compound of formula (I).
[0201] It will be understood by a person skilled in the art that
the term "free lyso-Gb1-sulfatide" as used herein preferably refers
to lyso-Gb1-sulfatide which is as such present in a sample from a
or the subject, such as blood including dried blood or plasma or
serum, and, preferably, is not the result of a manipulation of the
sample of said subject. Such manipulation of a sample can be the
one described by Groener et al. (Groener et al., Biochimica et
Biophysica Acta 1781(2908)72-78, 2007). In accordance therewith,
free lyso-Gb1-sulfatide which is present as such in the blood
including dried blood or plasma or serum of a subject from whom the
sample is taken, is more particularly not a lyso-Gb1-sulfatide
which is generated by chemical, biochemical or physical treatment
of the sample contained in the blood sample, plasma sample and/or
serum sample, preferably outside of the body of the patient.
Preferably, free lyso-Gb1-sulfatide is not a lyso-Gb1-sulfatide
prepared by chemical treatment of free lyso-Gb1-sulfatide as
contained in a sample of a subject, whereby the sample is
preferably a sample selected from the group consisting of plasma,
serum and blood including dried blood. More preferably, free
lyso-Gb1-sulfatide as used herein is different from
lyso-Gb1-sulfatide prepared by Toda K. et al (Toda K. et al.,
(1989) Biochemical and Biophysical Research Communications, Vol.
159, No. 2, pp 605-611); in other words, Toda K et al. prepared by
means of chemical treatment of lyso-Gb1-sulfatide free
lyso-Gb1-sulfatide without recognizing that such free
lyso-Gb1-sulfatide, although in a much lower amount, was already
present in the sample and more specifically a blood sample
including plasma sample serum sample and dried blood sample; by
subjecting lyso-Gb1-sulfatide to said chemical treatment, the
amount of free lyso-Gb1-sulfatide already present in the sample as
such, was obscured.
[0202] From the above it is evident that both Toda K et al. and
Groener et al. followed the same strategy in terms of
derivatization. It will be also understood by a person skilled in
the art that free lyso-Gb1-sulfatide as used herein, preferably is
present in addition to Gb1-sulfatide and is a compound produced by
the subject's metabolic activities. Accordingly, Gb1-sulfatide,
which is the molecule that is accumulated in connection with MLD is
present in the sample from the subject and has compared to the
molecule in a free lyso form, i.e. free-lyso-Gb1-sulfatide, present
in the blood, and plasma and/or the serum of the subject at least a
fatty acid moiety linked to the--primary--amino group of the
sphingosine moiety of lyso-Gb1-sulfatide.
[0203] In an embodiment of the biomarker according to the present
invention the biomarker is detected by means of immunoassay, mass
spectrometric analysis, biochip array, functional nucleic acids
and/or a fluorescent derivative of the biomarker. In connection
therewith it is important to note that such detection allows for
the selective detection of the biomarker as present in the blood of
a subject as such and particularly is not the result of a
manipulation of the sample of said subject resulting in a change of
the concentration of the biomarker, such as the derivatization of
Gb1 into lyso-Gb1 according to the method of the prior art as
described above. Such manipulation may result in the inability to
distinguish the biomarker which is present in its free-lyso-form
from the substance which is the result of said manipulation, for
example the result of said derivatization. Thus the biomarker of
the present invention cannot be detected as such and the level of
said biomarker cannot be determined as such, respectively, without
detecting the manipulated further substance, e.g. Gb1 converted
into lyso-Gb1 according to the method of the prior art. In the
light thereof it will be immediately understood that the biomarker
present in the blood of the subject such as free-lyso-Gb1-sulfatide
present as such in the blood of the subject, is also present in the
sample of the subject as such and may, nevertheless, be selectively
labeled with and/or linked to a means such as a fluorescent dye or
a nucleic acid molecule specifically binding the biomarker. Such
selective labeling or linking allows detecting and/or determining
the level of the labeled or linked biomarker, without labeling of,
linking to or converting a further substance, such as the converted
lyso-Gb1 of the prior art, which cannot be distinguished from the
biomarker, more precisely the labeled or linked biomarker. In
connection therewith, e.g. a fluorescent derivative of the
biomarker of the present invention concerns a biomarker which is
labeled with and/or bound to a fluorescence dye or molecule, i.e.
resulting in a fluorescent derivative of the biomarker, which
allows for detecting the fluorescent derivative and/or determining
the level of the fluorescent derivative of the biomarker of the
invention.
[0204] The term "sample" as used herein means preferably a limited
quantity of a subject's material, wherein said subject's material
is part of or has been taken from a subject and/or a subject's
body. Preferably, said material is selected from the group
comprising body fluids such as blood, a blood product such as
preferably plasma, serum, urine, saliva, cerebrospinal fluid and
lymph, as well as stool or any kind of tissue and or cell material
being part of a subject and/or a subject's body. A particular
preferred sample is a serum sample or a plasma sample which is used
or for use in any of the methods of the invention. A further
preferred sample is a dried blood sample. It will be acknowledged
by a person skilled in the art, that the presence of and/or a level
of a biomarker of the invention in said sample is intended to be
similar to and represent the presence and/or the level of the
biomarker in a larger amount of that subject's material. More
precisely and as an illustrative, non-limiting example, a level of
a biomarker of the invention determined in a sample of, e.g., some
ml of blood from a subject also represents a level of said
biomarker in the blood of the subject's body. Furthermore, in an
embodiment of the method of the invention for diagnosing MLD in a
subject, a sample from the subject comprises said subject's
material in a form, for example processed, fixed and/or preserved
such that said sample is suitable for use in the method of the
invention, whereby such processing, fixing and/or preserving
preferably does not generate lyso-Gb1-sulfatide which was not as
such present in the blood of the patient. The subject's material in
the sample may thus be diluted, for example with a solvent suitable
for the method of the invention such as methanol and/or water, may
be dried, for example on a filter card, may be resolved after
having been dried such, for example with a solvent suitable for the
method of the invention such as methanol and/or water, or a
substance may be added, wherein said substance prevents blood from
coagulation such as for example EDTA, citrate or heparin. It will
be further understood by a person skilled in the art that the
method of the invention comprises that said subject's material is
separated into single components of said subject's material and/or
single components of said subject's material are extracted from
said subject's material, for example blood is separated into plasma
or serum and cellular blood components or protein is precipitated
from the sample. Accordingly, in an embodiment of the method
according to the present invention wherein the method comprises
protein precipitation and/or HPLC, precipitation of protein
preferably results in a) a precipitation of cellular blood
components and/or protein, more preferably forming a pellet after a
step of centrifugation, and b) the biomarker being not precipitated
and being present in the supernatant after a step of
centrifugation. A person skilled in the art will immediately
understand that in an embodiment of the method according to the
present invention wherein the method comprises HPLC a supernatant
containing the biomarker(s) of the present invention or a part
thereof is subjected to HPLC. In connection therewith it is
important to understand that the supernatant or a part thereof
which is subjected to HPLC comprises the biomarker to be detected
as well as, preferably, an internal standard. In an embodiment of
the method of the invention wherein an internal standard is added
to the sample, the internal standard may be added to the sample
before or after a precipitation step, i.e. the internal standard
may be added into the sample immediately after the sample is taken
from the subject or after thawing of the sample such as the blood,
plasma or serum before analysis, or may be added to the supernatant
which is subjected to HPLC, as well as in between these time
points. A person skilled in the art will know, how and when an
internal standard is preferably added to the sample in order to
achieve an accurate detection and determination of a level of the
biomarker. It is within the present invention that the terms "dry
blood" and "dried blood" are preferably used in an interchangeable
manner, it not explicitly indicated to the contrary.
[0205] It will be immediately understood that after such
processing, fixing and/or preserving the sample is subjected to the
methods of the invention for detecting and/or determining the level
of a biomarker contained in said sample whereby such processing,
fixing and/or preserving preferably does not generate
lyso-Gb1-sulfatide which was not present in the sample from the
patient as such.
[0206] In an embodiment of the method of the present invention
wherein whole blood is collected on a dry blood filter card
preferably approximately 50 to 200 .mu.l of full blood, plasma or
serum are collected on a spot of said dry blood filter card having
a diameter of 3 mm. A person skilled in the art will acknowledge
that the exact volume thus collected may vary depending on the
hematocrit of the specific patient.
[0207] The levels of glucosylceramide and its precursor ceramide
were used in the prior art to correlate their presence in plasma
with the severity of Gaucher's disease type I and the response to
the application of therapy (Groener et al., Biochimica et
Biophysica Acta 1781(2908)72-78, 2007). Thereby, the level of lyso
glucosylceramide was found to be different although ceramide levels
were not significantly different in the plasma of treated and
untreated Gaucher's disease type I patients.
[0208] In the study reported by Groener et al. (Groener et al.,
supra) the ratio of glucosylceramide/ceramide was used to
discriminate between Gaucher's disease patients and healthy
patients. glucosylceramide and ceramide were measured with high
performance liquid chromatography (HPLC) essentially as described
in Groener et al. (J. E. M. Groener et al., Clin. Chern. 53 (2007)
742-747). In connection therewith it is important to understand
that glucosylceramide present in the plasma mainly consists of a
sugar moiety and a ceramide moiety. The ceramide moiety comprising
a sphingosine and a fatty acid moiety. According to the method of
the prior art lipids are extracted and ceramide and
glucosylceramide are deacetylated by alkaline hydrolysis thus
forming the lyso form, i.e. lyso-glucosylceramide (T. Taketomi et
al., J. Biochem. (Tokyo) 120 (1996) 573-579). Subsequently, the
thus produced lyso-glucosylceramide is labeled with a fluorescence
dye by derivatization with O-phthaldialdehyde (OPA) at the primary
amine group. Afterwards the derivatized sphingoid bases were
separated by reverse phase HPLC and detected with a fluorescence
detector. Thus said method of the prior art is able to detect total
glucosylceramide consisting of free lyso-glucosylceramide and
glucosylceramide and is not able to distinguish a level of free
lyso-glucosylceramide from a level of glucosylceramide in a sample
from a subject. The level of said total glucosylceramide after
cleavage of the various fatty acid moieties from the NH.sub.2-group
of the glucosylceramide is usually in a range of from 5 to 30 .mu.g
per mL plasma or serum. From this it is evident that in the method
of Groener et al. (Groener et al., supra) the total
glucosylceramide which can be prepared and obtained, respectively,
from a sample, preferably a blood sample, from a subject is used as
a biomarker rather than the free lyso-glucosylceramide contained in
the blood and accordingly also in the sample without performing a
cleavage of the fatty acid moiety/moieties, preferably a cleavage
performed by an operator handling the sample. Insofar, the present
invention is related to the detection of free lyso-Gb1-sulfatide
rather than total-Gb1-sulfatide.
[0209] Although total glucosylceramide measured as
lyso-glucosylceramide in said study of the prior art was increased
in plasma of said patients, said increase in total glucosylceramide
was not prominent and thus the specificity and the sensitivity of
the method were low showing that glucosylceramide is not suitable
as a biomarker for Gaucher's disease.
[0210] It is an embodiment of the methods of the present invention
comprising detecting and/or determining the level of free
lyso-Gb1-sulfatide in a sample from a subject that free
lyso-Gb1-sulfatide and/or the level of free lyso-Gb1-sulfatide is
determined separate from and/or apart from Gb1-sulfatide or a level
of Gb1-sulfatide which may be present in the blood of a subject. In
a further embodiment Gb1-sulfatide and/or a level of Gb1-sulfatide
is detected/determined in addition to the detection of and/or the
determining of a level of free lyso-Gb1-sulfatide.
[0211] Importantly, each primary amine circulating in the plasma
and being sufficiently lipophilic to be extracted concomitantly
with Gb1-sulfatide using an organic solvent according to said
method of the prior art is labeled accordingly and is thus able to
disturb the detection of cleaved lyso-Gb1-sulfatide.
[0212] In an embodiment of the biomarker according to the present
invention what has been outlined above with regard to free
lyso-Gb1-sulfatide also applies to any biomarker of the present
invention being present as in a free-lyso form.
[0213] Insofar, the biomarker of the present invention and uses
thereof clearly exceed the performance of methods for diagnosing
MLD known the prior art, more specifically, attempts of such
methods using biomarkers. It will be immediately understood that a
method for diagnosing MLD in accordance with Toda K et al. (Toda K
et al., supra) and thus analogous to the method applied by Groener
et al. for diagnosing Gaucher's disease (Groener et al., supra) is
prejudicial compared to the methods of the present invention in
that diagnosing of MLD based on such method of the prior art using
total Gb1-sulfatide rather than free lyso-Gb1-sulfatide as the
method of the prior art using total glucosylceramide rather than
free lyso-glucosylceramide is not suitable for reliable clinical
application thereof, i.e. the method has no sensitivity and
specificity sufficient to diagnose Gaucher's disease by a reliable
statistically secured prediction.
[0214] In clear contrast thereto the present invention provides
methods for the diagnosis of MLD and biomarkers used in said
methods which allow the diagnosis of MLD with high sensitivity and
high specificity.
[0215] The term "MLD status", also referred to herein as "MLD
status", preferably refers to the status of the disease in the
subject. Examples of types of MLD statuses include, but are not
limited to, the subject's risk of suffering or developing MLD, the
stage of the disease in a subject and the effectiveness of
treatment of the disease. Other statuses and degrees of each status
are known in the art. In an embodiment of the present invention the
MLD status comprises a severe, mild, or healthy MLD status.
[0216] The term "diagnosing" as preferably used herein, means
determining the presence or the absence of a disease or disorder in
a subject and/or determining whether a subject is at risk for
developing a disease, a disorder or symptoms related to a disease
or disorder as well as predicting a status of a disease.
"Diagnosis" or "diagnosing" as used herein also preferably means
that a cause of symptoms of a disease which are present or will be
present is identified.
[0217] In connection therewith it is important to note that a
person skilled in the art, such as a skilled clinician consulted by
a subject suffering from symptoms or suspected to be ill, applies
the methods of the present invention and thus determines whether a
subject is at risk for developing a disease, particularly MLD,
whether a subject suffers from such disease or predicts the status
of such disease, preferably based on the result obtained by the
practicing of the methods of the present invention.
[0218] Based on said diagnosis the person skilled in the art will
recommend to apply, maintain, reduce, elevate or not apply a
therapy or to perform further diagnostic tests.
[0219] It is thus an embodiment of the method of the present
invention for diagnosing MLD that the method comprises giving a
recommendation whether a therapy should be applied, maintained,
reduced, elevated or not applied.
[0220] The term "detecting" in the context of the present invention
preferably means a method which includes detecting the presence or
absence of a substance in a sample and/or qualifying the type of
said substance. Detecting can be accomplished by methods known in
the art and those further described herein, including, but not
limited to, the direct measurement of the affected protein(s) e.g.
the sequencing of the gene coding for ARSA. Any suitable method can
be used to detect one or more of the biomarkers described herein.
These methods include, without limitation, mass spectrometry (e.g.
HPLC-MS/MS), fluorescence (e.g. sandwich immunoassay),
HPLC-fluorescence or HPLC-UV preferably after derivatization of
free lyso-Gb1-sulfatide.
[0221] A biomarker as preferably used herein, is any biological
compound, such as a protein and a fragment thereof, a peptide, a
polypeptide, a proteoglycan, a glycoprotein, a lipoprotein, a
carbohydrate, a lipid, a nucleic acid, an organic or inorganic
chemical, a natural polymer, and a small molecule, which is
differentially present in a sample from a subject of one phenotypic
status (e.g. having a disease) as compared with another phenotypic
status (e.g. not having the disease) and which may be isolated
from, or measured in the sample from the subject. Furthermore, the
biomarker can be the entire intact molecule, or it can be a portion
thereof which is preferably detected by mass spectrometric
analysis, an antibody, another protein specifically binding the
biomarker, functional nucleic acids specifically binding the
biomarker and/or a fluorescent label. A biomarker is furthermore
considered to be informative if a measurable aspect of the
biomarker is associated with a given status of the patient, such as
a particular status of MLD. Such a measurable aspect may include,
for example, the presence, absence, or the level of the biomarker
in the sample from the subject and/or its presence as part of a
profile of biomarkers. A measurable aspect may also be a ratio of
two or more measurable aspects of biomarkers, which biomarkers may
or may not be of known identity, for example. A profile of
biomarkers comprises at least two such measurable aspects, where
the measurable aspects can correspond to the same or different
classes of biomarkers such as, for example, a nucleic acid and a
carbohydrate. A biomarker profile may also comprise at least three,
four, five, 10, 20, 30 or more measurable aspects. In one
embodiment, a biomarker profile comprises hundreds, or even
thousands, of measurable aspects. In another embodiment, the
biomarker profile comprises at least one measurable aspect of at
least one biomarker and at least one measurable aspect of at least
one internal standard.
[0222] In an embodiment of the method according to the present
invention an internal standard is added to a sample from a subject.
It will be acknowledged that by said addition of internal standard,
also referred to herein as IS, to the sample, i.e. spiking of the
sample, to be subjected to the method according to the present
invention, the concentration of IS in the sample is known and,
e.g., by determining the area under the peak, i.e. the peak area,
of the internal standard in, e.g., an HPLC-mass spectrometric
chromatogram the relation between a peak area and a concentration
of a substance, e.g. of IS and/or the biomarker of the present
invention, e.g. free lyso-Gb1-sulfatide, can thus be calculated,
e.g., by calculating the ratio of the peak area of free
lyso-Gb1-sulfatide and the peak area of IS. A person skilled in the
art will further acknowledge that various molecules may be used as
an IS. Nevertheless an IS having a similar chemical structure or an
isotopically labeled lyso-Gb1-sulfatide compared to the molecule
such as the biomarker, e.g. free lyso-Gb1-sulfatide, is preferable.
In accordance therewith, the present inventors have in an
embodiment chosen lyso-Gb2 which is not present as such in nature
in concentrations which could influence the precise determination
of lyso-Gb1-sulfatide. In a preferred embodiment the molecule being
the IS can be distinguished from the biomarker or the biomarkers of
the present invention, e.g. free lyso-Gb1-sulfatide, in the method
of the present invention. In a further preferred embodiment the IS
is selected such that a molecule which is ideally not present or
rare in nature. In an embodiment of the present invention where the
internal standard is added to a sample from a subject, it is
preferred that the IS is added such that it is dissolved in a
solvent, e.g. ethanol, prior to said addition to the sample. In a
further preferred embodiment that the solvent is selected such that
said solvent is capable of causing protein precipitation,
preferably is capable of causing the protein precipitation step as
subject to the method of the present invention.
[0223] In some embodiments of the present invention a protein
precipitation and/or protein precipitation step is part of the
method of the present invention. It will be understood that
precipitation as used herein, preferably means the formation of a
solid in a solution, i.e. for example the formation of a protein
precipitate in a sample, e.g. serum, from a subject. When
precipitation, e.g. protein precipitation, occurs in a sample, the
solid formed is called the precipitate, or when compacted by a
centrifuge, a pellet. The liquid remaining above the solid is in
either case called the supernatant. The present invention
contemplates different methods of precipitation and/or separating
said supernatant and said precipitate or pellet, comprising, among
others, settling or sedimentation and centrifugation. A person
skilled in the art will know further methods for protein
precipitation and/or for separating a supernatant and a protein
precipitate, nevertheless said skilled person will acknowledge that
if a method, preferably a method of the invention, is applied were
precipitated protein will disable a device such as a column or
HPLC-column used in connection with the present invention the
precipitated protein is preferably separated from the solvent
and/or the sample.
[0224] In some embodiments of the present invention a level of a
biomarker of the present invention, e.g. free lyso-Gb1-sulfatide,
determined by a method of the present invention in a sample is
compared to a level of the same or another biomarker of the present
invention determined by a method of the present invention in
another sample, e.g. from the same patient, from another patient,
from a control and/or from the same or different time points,
and/or a cut-off value, and/or a level of a control and/or a level
of an IS. In connection therewith "comparing" or "compared to" as
used herein, preferably means the mathematical comparison of the
two or more values of the levels of the biomarker(s). It will thus
be immediately evident whether one of said values is higher, lower
or identical if at least two of such values are compared with each
other.
[0225] The term "cut-off value" as preferably used herein refers to
a level, concentration and/or a titer of a biomarker of the present
invention, more preferably a level range, concentration range
and/or titer range of the biomarker.
[0226] In one particular embodiment thereof [0227] using free
lyso-Gb1-sulfatide as the biomarker allows for [0228] diagnosing
MLD using a cut-off value for free lyso-Gb1-sulfatide of 0.05 ng/ml
plasma or serum.
[0229] In a further particular embodiment thereof using free
lyso-Gb1-sulfatide as the biomarker allows for [0230] diagnosing
MLD using a cut-off value for free lyso-Gb1-sulfatide of 0.05 ng/ml
plasma or serum with a sensitivity of 100% and a specificity of
100%.
[0231] In some embodiments of the present invention the level of
the biomarker is also determined in a control. As used herein, a
control is preferably a sample from a subject wherein the MLD
status of said subject is known. In an embodiment a control is a
sample of a healthy patient. In a further embodiment an amount of
said biomarker is added to said sample of a healthy patient prior
to determining the level of said biomarker in said sample of a
healthy patient comprising said added biomarker with a method of
the present invention. In a further embodiment the control is a
sample from at least one subject having a known MLD status, such
known MLD status comprising severe, mild, or healthy MLD status,
e.g. a control patient. In a further preferred embodiment the MLD
status also comprises the genetic status with regard to mutations
of the gene or genes, affected in said disease, comprising the gene
coding for ARSA, i.e. comprising the subject having homozygous
and/or compound heterozygous mutations, the subject being a carrier
of a mutation.
[0232] In a further preferred embodiment the control is a sample
from a subject not being treated for MLD. In a still further
preferred embodiment the control is a sample from a single subject
or a pool of samples from different subjects and/or samples taken
from the subject(s) at different time points.
[0233] The term "level" or "level of a biomarker" as used herein,
preferably means the concentration of a substance and/or titer of a
substance, preferably of a biomarker of the invention and more
preferably of free lyso-Gb1-sulfatide, within a sample of a
subject. It will be understood by a skilled person that in certain
embodiments said sample is not necessarily subjected to a method of
the invention as a non-processed sample, the method comprising
determining a level of said biomarker, i.e. said sample may be
subjected, e.g. to a step of protein precipitation, separation,
e.g. centrifugation and/or HPLC and subsequently subjected to a
step of determining the level of the biomarker, e.g. using mass
spectrometric analysis. It should be further noted that whenever
the term "a" level of a biomarker is used in connection with a
level of the biomarker of the invention which is to be determined
according to the present invention, "the" level of the biomarker of
the present invention which is to be determined by the methods of
to the present invention and which is contained in the sample
subjected to the method(s) of the invention is meant. A preferred
sample is either a blood sample, a serum sample or a plasma
sample.
[0234] The level of a biomarker is different between different
statuses of MLD if the mean or median level of the biomarker in the
different groups is calculated to be statistically significant.
Common tests for statistical significance include, among others,
t-test, ANOVA, Wilcoxon, Mann-Whitney, odds ratio and
Kruskal-Wallis. Biomarkers, alone or in combination, provide
measures of relative risk that a subject belongs to one phenotypic
status or another. Therefore, biomarkers of the present invention
are useful in an embodiment of the present invention as markers for
disease, therapeutic effectiveness of a drug or a treatment.
[0235] The term "determining the level" of a biomarker as used
herein, preferably means methods which include quantifying an
amount of at least one substance in a sample from a subject and/or
quantifying an amount of said substance contained in a part of the
body of the subject, such as saliva, blood, lymph, serum, plasma or
liquor and/or quantifying an amount of said substance in the
subject, the substance being selected from the group comprising a
biomarker.
[0236] It will be understood by a person skilled in the art that
detecting and/or determining the level of free lyso-Gb1-sulfatide
in a sample from the subject, thus preferably comprises that
Gb1-sulfatide present in the blood of a subject is not chemically
converted, transformed or derivatized such that free
lyso-Gb1-sulfatide cannot be detected and/or the level thereof
cannot be determined separate from and/or apart from Gb1-sulfatide.
The person skilled in the art will acknowledge that Gb1-sulfatide
present in a sample from a subject which is subjected to a step of
deacylation, e.g. by hydrolysis in methanolic sodium hydroxide,
will result in cleavage of the fatty acid moiety from the
Gb1-sulfatide and thus will undesirably result in a chemically
converted, transformed or derivatized form of Gb1-sulfatide which
cannot be differentiated from free lyso-Gb1-sulfatide. It is thus
the merit of the present inventors to recognize that free
lyso-Gb1-sulfatide apart from Gb1-sulfatide is useful in a method
for diagnosing MLD.
[0237] In a preferred embodiment of the methods of the present
invention the method is for detecting and/or determining the level
of free lyso-Gb1-sulfatide in a sample from a subject, wherein
Gb1-sulfatide present in the sample from the subject is not
subjected to a step resulting in deacylation of Gb1-sulfatide,
preferably is not subjected to a step resulting in cleavage off of
a fatty acid moiety from the Gb1-sulfatide contained in the sample.
In a further preferred embodiment of the method of the present
invention Gb1-sulfatide present in the sample from the subject is
not chemically converted, transformed or derivatized. In a still
further preferred embodiment of the method of the present invention
free lyso-Gb1-sulfatide present in the sample from the subject is
separated from Gb1-sulfatide present in the sample from the subject
prior to a step that would result in cleavage of a fatty acid
moiety from the Gb1-sulfatide and/or prior to a step in which
Gb1-sulfatide is chemically converted, transformed or derivatized.
In a still further preferred embodiment a step of detecting and/or
determining the level of a biomarker in a sample from the subject,
wherein the biomarker is free lyso-Gb1-sulfatide, is performed
subsequent to separation using HPLC by application of mass
spectrometric analysis.
[0238] In an embodiment of the methods of the invention a subject
will be considered to be healthy regarding MLD if it has no
mutation of the functional parts of the gene coding for ARSA and/or
no mutation of the gene coding for ARSA resulting in a reduction of
or deficiency of the respective protein or the activity thereof,
resulting in symptoms associated with MLD.
[0239] A subject is considered to be a healthy subject with regard
to MLD if the subject does not suffer from symptoms associated with
MLD. Moreover in an embodiment of the methods of the invention a
subject will be considered to be healthy regarding MLD if it has no
mutation of the functional parts of the gene coding for ARSA and/or
no mutation of the gene coding for ARSA resulting in a reduction of
or deficiency of the respective proteins or the activity thereof,
resulting in symptoms associated with MLD.
[0240] In connection therewith it is important to understand that a
patient being a carrier of a mutation as outlined above is not
considered to be a healthy subject within the meaning of the
present invention although said carrier may not suffer from
symptoms associated with MLD. In certain embodiments of the methods
of the present invention MLD also comprises MLD carrier. It is
important to note that the methods of the invention are equally
suitable to identify an MLD carrier. The methods of the present
invention are suitable to diagnose whether or whether not a subject
is an MLD carrier. The method of the present invention is further
suitable for differentiating, diagnosing and/or differentially
diagnosing whether a subject is healthy, is an MLD carrier or is an
MLD patient.
[0241] Said mutations, i.e. mutations of the gene encoding ARSA,
will be detected if a sample from the subject is subjected to a
genetic testing for such mutations as described herein. In a
further embodiment of the present invention a sample from a healthy
subject is used as a control sample or as a blank matrix in the
methods of the present invention. A blank matrix as used herein is
preferably a sample from a healthy subject. Nevertheless it will be
understood that such a blank matrix may contain a native level of
free lyso-Gb1-sulfatide.
[0242] In an embodiment of the present invention the level of a
biomarker is indicative for the subject for suffering from or for
being at risk for developing a disease or disorder. The level of
the biomarker determined by the method according to the present
invention is compared to a control level of the biomarker, wherein
the result of said comparison allows for diagnosing a disease.
[0243] More specifically, comparing the level of the biomarker in
the sample from the subject to the control level of the biomarker
comprises comparing the level of the biomarker in the sample from
the subject to a cut-off value, wherein if a level of the biomarker
in the sample from the subject is higher than the cut-off value,
this is indicative that the subject is suffering from or is at risk
for developing MLD; and/or wherein if a level of the biomarker in
the sample from the subject is lower compared to the cut-off value
this is indicative that the subject is not suffering from or is not
at risk for developing MLD.
[0244] The term "being at risk for developing a disease" as used
herein preferably means that it is likely that a subject suffer
from said disease and/or will develop said disease or symptoms
associated with said disease, particularly if no treatment is
applied. In connection therewith it has to be acknowledged that
LSDs are genetic disorders and thus the occurrence of relatives,
particularly parents having said disease or having a mutation known
to be the cause of said disease are indicative for a subject, e.g.
the child of two MLD patients, to be at risk for developing said
disease. It will be furthermore acknowledged that the progression
of a disease is linked to the occurrence of symptoms as well as the
severity of said symptoms. Accordingly, a person not suffering from
symptoms at present, however, may be at risk for developing the
disease, for example, because although genetically mutations of a
gene, known to cause a disease are present, no symptoms or no
severe symptoms occur. Nevertheless, it will be immediately
understood that the methods and biomarkers of the present
invention, particularly if the level(s) of said biomarker(s)
according to the present invention are elevated, allow for
diagnosing that such subject is at risk for developing the disease
independent from the presence or absence of symptoms. Accordingly,
the methods according to the present invention allows for
determining whether a subject is at risk of suffering from MLD. It
is also within the present invention that a therapy is applied,
maintained, reduced, elevated or not applied based on whether the
subject is at risk of suffering from MLD or not.
[0245] It is also within the present invention that comparing the
level of the biomarker in the sample from the subject to a control
level allows for determining the severity of MLD, wherein if a
level of the biomarker in the sample from the subject is within the
cut-off value that is indicative that the subject is suffering from
or is at risk for developing MLD of a more severe status or
progression; and wherein if a level of the biomarker in the sample
from the subject is lower or higher compared to the control level,
i.e. the cut-off value, that is indicative that the subject is not
suffering from or is not at risk for developing MLD of a less
severe status or progression. In a further embodiment of the
present invention that comparing the level of the biomarker in the
sample from the subject to the control level comprises comparing a
level of the biomarker in said subject to a level of the biomarker
detected in a sample from a control, wherein if a level of the
biomarker in the sample from the subject is elevated, increased or
higher compared to the control sample this is indicative that the
subject is suffering from and/or is at risk for developing MLD;
and/or a level of the biomarker in the sample from the subject is
elevated, increased or higher compared to the control sample this
is indicative that the subject is suffering from or is at risk for
developing MLD of a more severe status or progression. Said control
preferably is selected from the group comprising healthy subjects,
subjects suffering from MLD or being at risk of suffering from MLD
symptoms, subjects being positively tested for a mutation or a
combination of mutations of the gene coding for ARSA, wherein the
mutation or the combination of mutations of the gene coding for
ARSA are indicative for a perspective of the subject to develop MLD
of a more severe or less severe status or progression. In a further
embodiment of the present invention that a control level is
determined in a sample from a control, wherein optionally free
lyso-Gb1-sulfatide is added to the sample from the control in a
specific quantity prior to determining the level of free
lyso-Gb1-sulfatide in the sample from the control.
[0246] It is the merit of the present inventors that a method for
diagnosing MLD in a subject could be established wherein the method
comprises detecting a biomarker in a sample from a subject, wherein
the biomarker is free lyso-Gb1-sulfatide, preferably further
comprising determining a level of the biomarker in the sample from
the subject, and more preferably further comprising comparing the
level of the biomarker in the sample from the subject to a cut-off
value, which shows high sensitivity, i.e. a sensitivity of at least
99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,
99.9% or 100%. In other words the sensitivity, which means the
proportion of actual positives which are correctly identified as
such is high, which means that the percentage of MLD patients
correctly identified as having the disease is as high as has been
outlined above. In contrast, in a statistic test as described
herein specificity means the proportion of negatives which are
correctly identified as negatives, in other words the percentage of
healthy patients correctly identified as not having MLD. A person
skilled in the art will acknowledge that thus an optimal prediction
of a diagnostic test such as in some embodiments of the methods
according to the present invention in general aims to achieve 100%
sensitivity, i.e. predict all patients having a disease, such as
MLD or being at risk of suffering from said disease, as having the
disease or being at risk from suffering from said disease,
respectively. Such sensitivity can be achieved with the cut-off
value for free lyso-Gb1-sulfatide being 0.05 ng/ml plasma or
serum
[0247] In an embodiment of the methods according to the present
invention a specificity of at least 80.0%, 85.0%, 90.0%, 95.0%,
97.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,
99.8%, 99.9% or 100% is preferred. In a further embodiment of the
present invention of the methods according to the present invention
the methods allow for diagnosing MLD in a subject independent from
a progression status of MLD in the subject. Such specificity can be
achieved with the cut-off value for free lyso-Gb1-sulfatide being
0.05 ng/ml plasma or serum.
[0248] More specifically, the methods of the present invention
allow for diagnosing MLD in a subject having an early status of MLD
as well as in a subject having an advanced or progressed status of
MLD.
[0249] The power of a method to correctly diagnose MLD is commonly
measured as the sensitivity of the method, the specificity of the
method or the area under a receiver operated characteristic curve
(also referred to herein as "ROC curve"). An ROC curve is a plot of
the true positive rate against the false positive rate for the
different possible cut-off values of a diagnostic method. An ROC
curve shows the relationship between sensitivity and specificity.
Sensitivity is the percentage of true positives that are predicted
by a test to be positive, while specificity is the percentage of
true negatives that are predicted by a test to be negative. An
ROC-curve provides the sensitivity of a test as a function of
1-specificity. The greater the area under the ROC-curve the more
powerful the predictive value of the test. Accordingly, an increase
in sensitivity will be accompanied by a decrease in specificity.
The closer the curve follows the left axis and then the top edge of
the ROC space, the more accurate the test. Conversely, the closer
the curve comes to the 45-degree diagonal of the ROC graph, the
less accurate the test. Therefore, the area under the ROC is a
measure of test accuracy. The accuracy of the test depends on how
well the test separates the group being tested into those with and
without the disease in question. An area under the curve (also
referred to herein as "AUC") of 1 represents a perfect method,
while an area of 0.5 represents a less useful method. Thus,
preferred diagnostic methods of the present invention have an AUC
greater then 0.50, more preferred methods have an AUC greater than
0.9 and most preferred methods have an AUC greater than 0.99.
[0250] Other useful and suitable measures for the utility of a
method are positive predictive value and negative predictive value.
A positive predictive value is the percentage of actual positives
that test as positive. A negative predictive value is the
percentage of actual negatives that test as negative.
[0251] A person skilled in the art will acknowledge that although
the specificity and/or the sensitivity of the methods according to
the present invention are as high as described above and were
determined as described in the Examples hereinafter, individual
cases may not be excluded where a patient having MLD will be tested
false negative or where a patient not having MLD will be tested
false positive with a method of the invention. A person skilled in
the art will thus immediately acknowledge that according to the
methods according to the present invention, wherein a level of a
biomarker is compared to a cut-off value and wherein said
comparison to said cut-off value is for use to diagnose a disease
such as MLD, said cut-off value represents a level of said
biomarker which discriminates a particularly disease status from
another, e.g. which discriminates a level of a biomarker indicative
that the subject has MLD from a level of a biomarker indicative
that the subject is an MLD carrier, and/or from a level and/or a
value in a healthy subject. Having said this, it is obvious for the
person skilled in the art that also according to the methods of the
present invention, wherein the method is for diagnosing MLD
individual cases may not be excluded where a patient having MLD
will be tested false negative or where a patient not having MLD
will be tested false positive, or where the type and/or status is
diagnosed incorrectly with a method of the invention.
[0252] Taking said cases into account while determining the
specificity and the sensitivity of the method according to the
present invention, the specificity and the sensitivity will be
lower than the above described values. Nevertheless, the person
skilled in the art will also acknowledge that such high specificity
and such high sensitivity as has been outlined above has never been
described before for a method for diagnosing MLD. Therefore it is
important to note that although the sensitivity and the specificity
of the method of the present invention may vary if patient
collectives other than the one reported in the Example part, e.g.
varying in number of patients, will are subject to the methods of
the present invention, it is the firm belief of the inventors that
no method known in the prior art using, especially using biomarkers
will achieve a higher specificity and a higher sensitivity compared
to the methods according to the present invention. This is
especially true since the limit of detection of the methods of the
present invention allows for determining the level of free
lyso-Gb1-sulfatide in healthy subjects. Accordingly, a diseased
subject tested false negative applying the methods of the present
invention is tested false negative for the reason that a level of
the biomarker in a sample from said false negative tested diseased
subject is as high as the level of the biomarker in a sample from a
healthy subject. In particular it is important to note that said
false negative tested subject is not tested negative for the reason
that the level of the biomarker was too low to be determined by the
method of the present invention.
[0253] A "limit of detection" or "limit of determination"--both
terms are used herein in a synonymous manner--of a substance such
as free lyso-Gb1-sulfatide, as used herein, preferably is a level
of the substance determined by a method for determining a level of
the substance, wherein a level less then or lower then said limit
of detection cannot be determined by said method. It is thus
immediately clear that a "cut-off value" and a "limit of
detection", as used herein, are preferably not necessarily
identical, although both reflect a certain level of a substance,
e.g. of a biomarker of the present invention. It will be
immediately understood that in contrast to a cut-off value will be
selected preferably such that selectivity and sensitivity of the
method are as high as possible. In contrast thereto a limit of
detection represents an absolute level of the biomarker of the
present invention which reflects the minimum level of biomarker
which can be detected with a method for determining the level of
said biomarker. It is thus immediately clear that a limit of
detection depends on the method for determining a level of a
substance and on the substance the level of which is to be
determined by the method. A skilled person will immediately
understand that a high limit of detection, e.g. higher than an
ideal cut-off value would possibly result in a low sensitivity of
the method since the percentage of true positives that are
predicted by a test to be positive also depends on whether a level
of the biomarker may be determined for said true positives. In
other words, if the limit of detection is higher than an ideal
cut-off value, true positives having a level of the biomarker
slightly higher than the cut-off value may not be distinguished
from true negatives having a level of the biomarker lower than the
cut-off value since no level of the biomarker may be determined for
both true positives having a level of the biomarker slightly higher
than the cut-off value and negatives having a level of the
biomarker lower than the cut-off value. It is thus immediately
clear that a low limit of detection is of advantage. It is
therefore also the merit of the inventors to show that a lower
limit of detection allows for a method for diagnosing MLD in a
subject comprising a step of determining a level of a biomarker
present in the sample with higher selectivity and sensitivity. An
"ideal cut-off value" as used herein, preferably is the cut-off
value as described herein the method using said ideal cut-off value
has the highest selectivity and sensitivity.
[0254] It is an embodiment of the methods according to the present
invention to comprise a step of validating said method by
diagnosing a disease or disorder, preferably MLD in a subject by
the method of the present invention; a step of diagnosing the
disease or disorder, preferably MLD, in a subject by a genetic
testing, comprising sequencing of a gene, preferably sequencing of
a gene a mutation of which is known to the one skilled in the art
to cause the disease or disorder, more preferably sequencing the
gene coding for ARSA in case of MLD; and comparing the results of
said method and said genetic testing. A healthy subject as used
herein, preferably is considered to be healthy with regard to a
disease or disorder if said subject is not suffering from symptoms
associated with said disease or disorder and if the result of a
genetic testing reveals no mutations of a gene a mutation of which
is known to the one skilled in the art to cause the disease or
disorder. A healthy subject also is understood to be a subject
being positively tested for not having MLD. In a preferred
embodiment a healthy subject is a subject not being a carrier of
MLD.
[0255] The term "qualifying MLD status" in a subject as used
herein, preferably means a classification of a subject's biomarker
profile selected from the group comprising to identify or detect
the presence or absence of MLD in the subject, to predict the onset
of or the risk for developing of MLD in the subject, to determine
the course of MLD in a subject, to determine and/or predict the
severity of MLD in a subject, to determine whether a subject
suffers from an early status of MLD or an advanced or progressed
status of MLD or to determine whether a level of a biomarker in a
subject has significantly changed over time.
[0256] The term "managing subject treatment" or "subject
management" as used herein, preferably refers to the behavior of
the clinician or physician subsequent to the determination of MLD
status. For example, if the result of the method according to the
present invention is inconclusive or there is reason that
confirmation of status is necessary, the physician may order new
tests, such as testing for the function of the affected proteins
and/or sequencing of the gene coding for ARSA. Alternatively, if
the status indicates that treating for MLD is appropriate, the
physician may schedule the subject for treating for MLD. Likewise,
if the status is negative or if the results show that treatment has
been successful, no further management may be necessary.
Nevertheless a person skilled in the art will immediately
acknowledge that besides gene therapy any therapy applied has to be
applied lifelong to an MLD patient. Furthermore it is an embodiment
of the present invention that managing subject treatment comprises
titrating of a dose of a drug applied as a treatment for MLD, e.g.
units of recombinant enzyme applied in ERT, administered to a
patient. In some embodiments of the methods of the present
invention wherein a level of a biomarker present in a sample from a
subject is determined at several points in time, or is compared to
other levels of the biomarker, a cut-off value and/or a level of
said biomarker in a control, a skilled person will apply or not
apply a therapy, or amend a therapy already applied in order to
treat or not to treat, or to continue treating MLD.
[0257] It is within the present invention that a skilled person
will apply a dosage and/or maintain a dosage or amend a dosage,
e.g. apply a dosage or a higher dosage, i.e. elevate a dosage, if
such a comparison of the level of a biomarker shows e.g. that the
level of said biomarker is higher than for example, a cut-off
value, i.e. the patient is diagnosed to have MLD; or that a level
determined in the same patient earlier in time is lower or the
same, i.e. a therapy applied is not sufficient, i.e. does not
result in a decrease in the level. On the other hand skilled person
will apply or not apply a dosage or maintain or reduce a dosage,
e.g. apply no dosage or a lower dosage, i.e. decrease a dosage, if
such a comparison of the level of a biomarker shows e.g. that the
level of said biomarker is lower than for example, a cut-off value,
i.e. the patient is diagnosed not to have MLD disease; or that a
level determined in the same patient earlier in time is higher,
i.e. a therapy applied is sufficient, i.e. does result in a
decrease in the level. In an embodiment of the present invention a
relatively high level of free lyso-Gb1-sulfatide based on such a
comparison is indicative for applying a high dosage of recombinant
enzyme applied in ERT and/or a relatively low level of free
lyso-Gb1 based on such a comparison is indicative for applying a
low dosage of recombinant enzyme applied in ERT. Nevertheless it
will also be immediately understood that a skilled person will
consider a patient's history, i.e. a skilled person managing
subject treatment of a patient suffering from MLD and being treated
such that a level of biomarker is lower than a cut-off value, for
example, will not decide to stop treatment rather than decrease a
dosage and increase the time between further applications of the
methods of the present invention.
[0258] The course of MLD may be determined by the method according
to the present invention by determining a level of the biomarker in
the sample from the subject at different time points in the course
of the disease. It is important to note that a single application
of a method for diagnosing MLD according to the present invention
allows for diagnosing MLD and in certain embodiments comprises a
step of managing subject treatment based on the diagnosis of
whether the subject is suffering from or for being at risk for
developing MLD. If a subject a sample of which is thus subjected to
the method of the present invention is tested positive for
suffering from or to be at risk for developing MLD a skilled
clinician will know how to decide concerning managing subject
treatment, i.e. how the subject will be treated, e.g. applying a
certain dose of enzyme in relation to an ERT. It will be
immediately understood that independent of the decision of a
skilled clinician on how to manage subject treatment the skilled
clinician may decide for at least one additional application of the
method according to the present invention on a later time point. It
is thus an embodiment of the present invention that the levels of
the biomarker determined at the different time points, wherein
different time points means at least two time points, may be
compared. Without wishing to be bound by any theory the present
inventors have found that the level of the biomarker of the present
invention in samples form one particular patient may be correlated
to the severity of the disease in said patient at the time point
the sample from the patient is taken. It will be thus immediately
understood that an elevated level of the biomarker determined in
the sample of a later time point compared to the level of the
biomarker determined in the sample of an earlier time point is
indicative for a more severe status of the subject at the later
time point compared to the status of the subject at the earlier
time point. A decreased level of the biomarker determined in the
sample of a later time point compared to the level of the biomarker
determined in the sample of an earlier time point is indicative for
a less severe status of the subject at the later time point
compared to the status of the subject at the earlier time point.
Accordingly, in one aspect the present invention provides a method
for determining the course of MLD in a subject comprising the step
of determining at several points in time a level of a biomarker
present in a sample from the subject, wherein the biomarker is free
lyso-Gb1-sulfatide In a further aspect the invention concerns a
method for determining the effectiveness of at least one treatment
applied to a subject being positively tested for suffering from or
being at risk for developing MLD comprising the step of determining
at several points in time a level of a biomarker present in a
sample from the subject, wherein the biomarker is free
lyso-Gb1-sulfatide. It will be immediately understood by a person
skilled in the art that the methods of the present invention thus
allow for selecting a therapy and/or adjusting the doses and/or
dosage of a selected therapy based on the results of the method of
the invention. If for example the subject is scheduled for treating
for MLD the method for diagnosing MLD in a subject according to the
present invention may be applied every 3 months and levels of the
biomarker thus determined will be compared in order to determine
the effectiveness of the treatment(s) and/or therapy/therapies
applied to the subject. If the subject reaches a status, wherein a
stable level of the biomarker is maintained over time the frequency
of application of the method for diagnosing MLD in a subject
according to the present invention may be reduced to every 6 month.
If the dosage of the therapy is changed, e.g. the units of
recombinant enzyme applied in ERT are reduced or increased, the
frequency of application of the method for diagnosing MLD in a
subject according to the present invention may be set back to every
3 month. By comparison of the determined levels of the biomarker in
the samples from the subject the skilled physician will recognize
whether the level of the biomarker increases, decreases or whether
a stable level of the biomarker is maintained over time.
Accordingly, the skilled physician may decide to reduce the dosage
of the therapy, e.g. the units of recombinant enzyme applied in
ERT; to increase the dosage of the therapy; or to maintain the
dosage of the therapy according to the comparison of the levels of
the biomarker determined with the method according to the present
invention. A significant reduction of the level of free
lyso-Gb1-sulfatide within a period of 12 month is indicative for a
successful therapy for MLD, wherein reduction as used herein,
preferably means that the level of free lyso-Gb1-sulfatide
determined by the method of the present invention determined at the
end of a time period is compared to the level of free
lyso-Gb1-sulfatide determined by the method of the present
invention determined at the beginning of said time period.
Accordingly the skilled physician may decide to reduce the dosage
of the applied therapy or to maintain the dosage of the therapy. If
the reduction of the level of free lyso-Gb1-sulfatide is
significantly weaker the skilled physician may decide to increase
the dosage of the therapy. It is also a merit of the present
inventors to have recognized that the reduction of the level of
free lyso-Gb1-sulfatide correlates with the effectiveness of a
therapy. The stronger the reduction of the level of the free
lyso-Gb1-sulfatide within a time period, e.g. 12 months, the more
successful is a therapy, such as for example ERT, SRT or a
chaperone based therapy. It is thus a further embodiment of the
present invention that the method of the present invention is for
comparing the effectiveness of a therapy or of at least two
therapies applied to a subject.
[0259] A person skilled in the art thus will acknowledge that the
progression, i.e. course of MLD, as well as the effectiveness of a
therapy in a single subject can be monitored by frequent
determining of the level of free lyso-Gb1-sulfatide in samples from
the subject.
[0260] In a further aspect the invention concerns a method for
determining the effectiveness of at least one treatment applied to
a subject being positively tested for suffering from or being at
risk for developing MLD comprising the step of determining at
several points in time a level of a biomarker present in a sample
from the subject, wherein the biomarker is free lyso-Gb1-sulfatide
In connection with what has been outlined above in relation to
managing subject treatment a person skilled in the art will
immediately understand that the effectiveness of one treatment or
the combination of at least two treatments may be compared applying
the methods of the present invention. Thus it is possible to test
and compare several new drugs, dosage forms, dosages or treatments
for MLD by the method of the present invention.
[0261] It is an embodiment of the present invention that the method
for diagnosing MLD according to the present invention is
independent of whether the subject has or has not been previously
treated for MLD. Thus the sample from the subject may be a sample
from a subject who has been previously treated for MLD as well as a
sample from a subject who has not been previously treated for MLD.
It is thus a further embodiment of the present invention that the
method of the present invention comprises a step of managing
subject treatment and/or determining a level of the biomarker in
the sample from the subject after subject management. Said subject
treatment can be based on the diagnosis of whether the subject is
suffering from or for being at risk for developing MLD; on the
detection of the biomarker in a sample from the subject after
subject management; or on the determining of the level of the
biomarker in the sample from the subject after subject management.
Nevertheless a person skilled in the art will understand that a
sample of some patients not having MLD or of some patients being
successfully treated for MLD will show a level of free
lyso-Gb1-sulfatide lower than the limit of detection.
[0262] Without wishing to be bound by any theory the present
inventors assume that the level of free lyso-Gb1-sulfatide present
in a sample from a subject further correlates with the severity of
the disease in a subject suffering from MLD. In connection
therewith the present inventors found by evaluating the results
provided herein (e.g. shown in FIG. 2 herein) assume that although,
in principle, the level of free lyso-Gb1-sulfatide is different in
particular individuals, and more specifically may be different in
particular individuals having the same mutation(s), that the higher
is a level of free lyso-Gb1-sulfatide, the higher is the severity
of a course of MLD in terms of a statistical mean according to a
clinical score. Thereby the level of free lyso-Gb1-sulfatide
correlates with the severity of MLD in that in patients being
positively tested for distinct mutations of the gene coding for
ARSA being known to generally causes a mild or a more severe course
of MLD, a level of free lyso-Gb1-sulfatide determined in said
patients statistically correlates with the severity generally
related to such mutation.
[0263] Thus a further embodiment of the different aspects of the
present invention concerns a method for determining the severity of
MLD in a subject comprising a step of [0264] a) determining a level
of the biomarker present in a sample from the subject wherein the
biomarker is free lyso-Gb1-sulfatide and a step of [0265] b)
determining the severity of MLD, e.g. by comparing the level of
free lyso-Gb1-sulfatide in a subject preferably determined by a
method of the present invention to a clinical score.
[0266] In connection therewith it is important to note that if a
level of free lyso-Gb1-sulfatide is determined in samples from the
patients suffering from MLD showing the L444 Pa mutation usually
linked to a more severe course of MLD upon sequencing of the
respective gene (homozygous and compound heterozygous) subjected to
a method of the present invention a mean-level of free
lyso-Gb1-sulfatide is higher than the mean-level of the free
lyso-Gb1-sulfatide determined in samples from the patients
suffering from MLD showing a mutation usually linked to a more mild
course of MLD upon sequencing of the respective gene, applying the
same method. A "mutation usually linked to a more severe course of
MLD" as used herein preferably is known to cause a more severe
course of MLD--this is especially true in case the subject is
homozygous as to said mutation. Corresponding to that in an
embodiment a higher mean-level of free lyso-Gb1-sulfatide is
determined in the homozygous compared to the homozygous mutation
usually linked to a milder course of MLD. Moreover patients having
a compound heterozygous usually linked to a more severe course of
MLD have a significantly lower free lyso-Gb1-sulfatide level, than
homozygous ones. A person skilled in the art will know clinical
scores to categorize the severity of MLD or symptoms or an entirety
of symptoms thereof. It is thus an embodiment of the method of the
present invention that the course of MLD in a patient is predicted
and more particularly the severity of MLD is determined based on
the level of the biomarker determined according to the method of
the present invention.
[0267] A person skilled in the art will acknowledge that a level of
the biomarker of the present invention determined in a sample from
a subject wherein said level of the biomarker is correlated with
the severity of MLD as described above, will be indicative for
applying a certain therapy and/or dose or dosage of said therapy.
For example, if the level of the biomarker determined according to
the methods of the invention is correlated with "severe" MLD status
the subject is scheduled for treatment of MLD and the method for
diagnosing MLD in a subject according to the present invention may
be applied every 3 months and levels of the biomarker thus
determined will be compared in order to determine the effectiveness
of the treatment(s) and/or therapy/therapies applied to the
subject. If the subject reaches a status, wherein the level of the
biomarker is correlated with a "mild" MLD or wherein a stable level
of the biomarker is maintained over time the frequency of
application of the method for diagnosing MLD in a subject according
to the present invention may be reduced to every 6 month.
[0268] In another aspect the present invention is related to a
method of determining the effectiveness of a composition for the
treatment of MLD. Such method may comprise the steps of determining
a level of free lyso-Gb1-sulfatide in a subject having MLD;
administering to said subject said compound in an amount sufficient
to determine the effectiveness of said compound; re-determining the
level of free lyso-Gb1-sulfatide in said subject; comparing the
level of free lyso-Gb1-sulfatide determined before and after
administering said composition, wherein a lower level of free
lyso-Gb1-sulfatide determined after administering said composition
compared to the level of free lyso-Gb1-sulfatide determined after
administering said composition indicates the effectiveness of said
compound for treating MLD.
[0269] MLD affects mostly children and they often die at a young
and unpredictable age, many within a few months or years of birth.
Many other children die of this disease following years of
suffering from various symptoms of their disorder.
[0270] A preferable biomarker for the diagnosis of MLD would allow
for diagnosis of MLD with high sensitivity and high specificity
independent from the age of the subject.
[0271] It is the merit of the present inventors having found that
the biomarkers of the present invention are useful for the
diagnosis of MLD in a subject independent from the age of the
subject. It is thus an embodiment of the present invention that the
method of the present invention allows for diagnosing MLD in a
subject independent from age, preferably under the provio that the
subject is at least 2 months old. In a preferred embodiment the
method of the present invention the subject is a subject of young
age. A subject of young age as used herein preferably is a subject
of less than 30 years of age, more preferably of less than 20 years
of age and most preferably of less than 10 years of age.
[0272] It will be acknowledged by a person skilled in the art that
the sensitivity and specificity of the diagnostic method of the
invention, i.e. the proportion of actual positives which are
correctly identified, depends at least to a certain extent on the
patient group tested. In connection with the instant invention and
the various methods of the invention, a patient or subject is
preferably a human of at least 2 months age.
[0273] It is within the invention that the methods of the invention
and in particular the method of the invention for diagnosing
metachromatic leucodystrophy is one for diagnosing juvenile form of
metachromatic leucodystrophy and/or adult form of metachromatic
leucodystrophy.
[0274] It will also be acknowledged by a person skilled in the art
that the cut-off value for free lyso-Gb1-sulfatide of 0.05 ng/ml
plasma or serum is one which is preferably applied in case the
limit of determination, which is also referred to herein as limit
of detection, of the analytical method used in determining the
level or concentration of free lyso-Gb1-sulfatide is 0.05 ng free
lyso-Gb1-sulfatide/ml plasma or serum, or in case the limit of
determination of the analytical method used in determining the
level or concentration of free lyso-Gb1-sulfatide is set to 0.05 ng
free lyso-Gb1-sulfatide/ml plasma or serum.
[0275] The present invention is now further illustrated by the
following figures and examples from which further features,
embodiments and advantages may be taken.
[0276] More specifically,
[0277] FIG. 1A is an HPLC-mass spectrometric chromatogram
displaying peak intensity of free lyso-Gb1-sulfatide and IS of a
healthy subject (upper chromatogram for free lyso-Gb1-sulfatide and
lower chromatogram for the internal standard);
[0278] FIG. 1B is an HPLC-mass spectrometric chromatogram
displaying peak intensity of free lyso-Gb1-sulfatide and IS of
subject diagnosed as MLD-positive (upper chromatogram for free
lyso-Gb1-sulfatide and lower chromatogram for the internal
standard);
[0279] FIG. 2 is a boxplot indicating levels in a first cohort of
MLD diagnosed subjects of free lyso Gb1-sulfatide in ng/ml plasma
compared to the levels in subjects positively diagnosed as
suffering from other LSDs of free lyso Gb1-sulfatide in ng/ml
plasma;
[0280] FIG. 3 is a boxplot indicating levels in a second cohort of
MLD diagnosed subjects of free lyso Gb1-sulfatide in ng/ml plasma
compared to the levels in subjects positively diagnosed as
suffering from other LSDs of free lyso Gb1-sulfatide in ng/ml
plasma, in subjects positively diagnosed as being MLD carrier of
free lyso Gb1-sulfatide in ng/ml plasma, and in subjects being
healthy controls;
[0281] FIG. 4 is a boxplot indicating levels of enzymatic
arylsulfatase A activity in U/mg protein in the second cohort of
MLD diagnosed subjects compared to the levels in subjects
positively diagnosed as suffering from other LSDs, in subjects
positively diagnosed as being MLD carrier, and in subjects being
healthy controls; and
[0282] FIG. 5 is a graph showing receiver operating characteristics
(ROC) for the diagnosis of MLD.
EXAMPLES
[0283] In the Examples described in the following human plasma or
serum was used as a sample from a subject. Nevertheless a person
skilled in the art will acknowledge that depending on the used type
of sample from a subject, e.g. comprising saliva, liquor, plasma,
serum, full blood, blood on a dry blood filter card or another
blood product, the method of the present invention has to be
adjusted to the type of sample and furthermore a cut-off value has
to be determined for each type of sample according to the method
described in the following examples. The present inventors have
found that using a sample of human serum in the method as described
below instead of a sample of human plasma will lead to identical
results in terms of the level of free lyso-Gb1-sulfatide, if the
sample of human serum and the sample of human plasma are taken from
the same subject, at the same time point, and if the samples were
measured in parallel; and, more particularly, will lead to the same
cut-off value.
Example 1: Method for the Detection of Free Lyso-Gb1-Sulfatide in
Human Plasma/Serum
[0284] Equipment
[0285] For detecting free lyso-Gb1-sulfatide in a sample of plasma
from a subject the following equipment was used.
TABLE-US-00001 Apparatus/Piece of Equipment Type/Producer HPLC pump
Series 200, Perkin Elmer, USA Sample injector Series 200, Perkin
Elmer, USA Column oven Series 200, Perkin Elmer, USA Mass selective
detector API 5000, AB SCIEX, USA/Canada Multi-tube vortexer
DVX-2500 Henry Troemner LLC, USA Vortex mixer Vortex Genie 2;
Scientific Industries, USA Centrifuge Megafuge 1.0; Heraeus,
Germany Multipette(s), pipette(s) Eppendorf, Germany Water bath
SW21-C, Julabo, Germany
[0286] Reagents
[0287] For detecting free lyso-Gb1-sulfatide in a sample of plasma
from a subject the following reagents were used.
[0288] To that extent that values depend on temperature (e.g. the
pH value) such values were determined at a temperature of
25.degree. C.
TABLE-US-00002 Reagent Purity Acetonitrile (ACN) HPLC-grade or
Gradient grade Acetone 99.5% Formic acid (FA) p.a., 98-100%
Methanol (MeOH) Gradient (LiChrosolv) Trifluoroacetic acid (TFA)
purum >98% Water ASTM-I
[0289] The abbreviation "p.a." as used herein means "pro
analysis".
[0290] The term "purum" as used herein, preferably means a
commercial grade of a chemical compound having a purity of the
above specified value.
[0291] ASTM-I as used herein refers to a water grade standard
purity achieved by purification methods comprising Reverse Osmosis
and Ultraviolet (UV) Oxidation.
[0292] Preparation of Calibration Standards
[0293] A lyso-Gb1-sulfatide stock solution was prepared dissolving
0.62 mg lyso-Gb1-sulfatide (as delivered by Matreya) in 5 mL of 50%
MeOH.
[0294] Subsequently the solution V1-A-626 was prepared as a mixture
of 50 .mu.L of lyso-Gb1-sulfatide stock solution and 10 mL 50% MeOH
as displayed in the following:
TABLE-US-00003 Label of exp. Volume of volume of resulting conc.
solution solvent solution [.mu.g/mL] [.mu.L] solution [mL] solvent
V1-A-626 0.60760 50 lyso-Gb1- 10 50% sulfatide- MeOH stock
[0295] Subsequently the Calibration Standards were prepared by
spiking solution V1-A-626 or higher concentrated Calibration
Standards into blank human plasma from healthy people.
[0296] A detailed spiking scheme will be displayed in the
following.
TABLE-US-00004 Label of resulting concentration Volume of volume of
solution [ng/mL] solution [.mu.L] solution solvent [mL] solvent
Volume [ml] Std4B-626 5.0016 49.8 V1-A-626 6 human 6.0498 plasma
Std3B-626 1.0003 1250 Std4B-626 5 human 6.25 plasma Std2B-626
0.19976 208 Std4B-626 5 human 5.208 plasma Std1B-626 0.050011 50.5
Std4B-626 5 human 5.0505 plasma
[0297] For calibration, calibration standards having four
concentration levels between 0.05 and 5.00 ng/mL were used, namely
Calibration Standards Std1B-626, Std2B-626, Std3B-626 and
Std4B-626.
[0298] Preparation of Control Samples
[0299] Control samples were prepared by spiking solution V1-A-626
or a higher concentrated control sample into a blank human
plasma.
[0300] A detailed spiking scheme will be displayed in the
following.
TABLE-US-00005 Volume volume Label of concen- of of blank resulting
tration solution matrix Volume solution [ng/mL] [.mu.L] solution
[mL] [m] QC-A2- 0.30032 556 QC-B7- 5 5.556 626 626 QC-B7- 3.0010
27.3 V1-A- 5.5 5.5273 626 626
[0301] Blank Matrix
[0302] As a blank matrix, human plasma of a healthy subject was
used containing no detectable levels of free lyso-Gb1-sulfatide.
Before using such blank matrix for spiking lyso-Gb1-sulfatide
should be determined in this matrix to assure the absence of
it.
[0303] Study Samples
[0304] Preparation of Internal Standard
[0305] The Internal Standard (IS1) stock solution was prepared
dissolving 1.00 mg of lyso-Gb2 (as delivered by Matreya) in 2 mL of
DMSO/MeOH (1/1; vol/vol).
[0306] Subsequently the Internal Standard Working Solution was
prepared as a mixture of 82 .mu.L of IS1 stock solution and 500 mL
of ethanol. The ethanol may be purchased from any commercial
source, wherein the ethanol is absolute ethanol having a grade
suitable for the methods described herein. A person skilled in the
art will recognize that proteins contained in 50 .mu.l of a sample
have to precipitate if 100 .mu.L of said Internal Standard working
solution are added to the sample.
[0307] Storing of Samples and Solutions
[0308] Control samples or study samples either were immediately
stored below -20.degree. C. at once or aliquots were transferred
into new glass vials before storing under the same conditions.
[0309] Concentrated solutions (stock solutions, V1-A-534 etc.) as
well as Internal Standard stock solutions were frozen below
-20.degree. C. pending next spiking.
[0310] Internal Standard working solution was stored below
-20.degree. C. until use.
[0311] The present inventors have found that free
lyso-Gb1-sulfatide is stable in the above mentioned solutions.
lyso-Gb1-Sulfatide is also stable at least for several weeks of
storage in plasma/serum below -20.degree. C. and over several
freeze/thaw cycles in plasma/serum.
[0312] Sample Preparation for Analysis
[0313] All samples used in an analytical batch are prepared for
analysis as follows: [0314] Frozen samples were thawed at
approximately 20 to 25.degree. C. in a water bath taking from
ambient conditions. After thawing the samples were mixed. [0315] 50
.mu.L of the sample were transferred into a sample vial [0316] 100
.mu.L of Internal Standard working solution (in EtOH) was added to
the sample [0317] The thus obtained mixture was subsequently mixed
using a DVX-2500 Multi-tube vortex device at 2500 rpm for about 30
seconds [0318] The thus obtained mixture was centrifuged for phase
separation at 4000 rpm for 2 minutes. [0319] Transfer of a volume
of the supernatant adequate to injection purposes (approx. 100
.mu.L) into appropriate (conical) auto-sampler vials
[0320] Methods
[0321] Chromatographic and Auto-Sampler Parameters
[0322] The samples prepared for analysis as described above were
subsequently subjected to the method described in the
following:
TABLE-US-00006 Parameter Scheduled range/description Mobile phase
solvent A 50 mM FA in water Mobile phase solvent B 50 mM FA in
ACN/acetone (1:1; vol/vol) Chromatographic run 0.0-0.5 min
isocratic: 10% B 0.5-0.6 min step gradient: 10% B .fwdarw. 33% B
0.6-4.7 min linear gradient: 33% B .fwdarw. 69% B 4.7-5.4 min
isocratic: 100% B 5.4-6.0 min isocratic: 10% B Flow 1.1 mL/min
Injection volume 15 .mu.L Injector flush 0.1% TFA in 70% MeOH
Column + Precolumn YMC Pro C8, 100 .times. 3 mm ID, 3 .mu.m Column
temperature 60.degree. C. Retention time approx. 4.3 to 4.9 min:
lyso-Gb1-sulfatide approx. 2.6 to 3.3 min: lyso-Gb 2 (IS)
[0323] The YMC Pro C8 column (Nr. 0S12S031003QT) used herein has
been purchased from YMC, Germany.
[0324] It will be appreciated by a person skilled in the art that
parameters where a ".+-." range is indicated represent parameters
which may be adjusted between sequences. A sequence as used herein,
preferably is a batch of defined numbers of samples, preferably 150
in maximum analyzed sequentially, wherein parameters comprising
flow and temperature remain unchanged. Adjustments and calibrations
performed between sequences are known to those skilled in the art
and comprise exchange of the column.
[0325] These adjustments within the specified limits are minor
changes and are recorded within the raw data of the study at the
measuring station.
[0326] Detection
[0327] The thus prepared samples were subsequently subjected to the
detection method the parameters of which are described in the
following:
TABLE-US-00007 MS Ioisation mode: Electrospray Ionisation (ESI) MS
polarity: positive MS detection mode: Multiple reaction monitoring
(MRM) Vaporizer temperature: 550.degree. C. .+-. 50.degree. C.
Ionisation voltage 5.5 kV Collisionally activated Pressure = 5 psi
dissociation (CAD) gas: Gas 1: Pressure = 57 psi Gas 2: Pressure =
67 psi Curtain gas: pressure = 39 psi Lateral position: 5 units
Vertical position: 4 units Quadrupole resolution unit .fwdarw. unit
Transitions 542.4 .fwdarw. 282.2 m/z lyso-Gb1-sulfatide (preferred
quantifier) 542.4 .fwdarw. 462.4 m/z lyso-Gb1-sulfatide (qualifier)
624.5 .fwdarw. 282.2 m/z lyso-Gb2 (Internal Standard) DP
(declustering potential) 50 V
[0328] A person skilled in the art will acknowledge that methods
for detecting free lyso-Gb1-sulfatide and/or determining the level
of free lyso-Gb1-sulfatide in a sample from a subject using mass
spectrometric analysis may also employ other transitions and
fragments which allow for specific detection of and/or
quantification of free lyso-Gb1-sulfatide in said sample from a
subject.
[0329] Evaluation and Calculation of Results
[0330] To evaluate and to calculate results obtained with the above
specified methods the following protocol were applied.
[0331] Rounding Procedure
[0332] Concentration data fed into and retrieved from the
chromatographic data system (CDS) were rounded to five significant
digits. Further calculations in the spreadsheet were performed to
full computational accuracy and subsequently rounded to the
significant digits/decimal places to be reported. Hence, deviations
of intermediate results might occur caused by rounding. Accuracy
and coefficients of variation (CV) will be reported with one and
two decimal places, respectively.
[0333] Note Referring to the Rounding Procedure:
[0334] The last digit reported would be up-rounded if the
subsequent digit was equal or greater than "5".
[0335] Regression and Statistics
[0336] Based on Calibration Standards the calibration curve fitting
were established using the data processing software by means of
peak area ratios (peak area of free lyso-substance contained in the
sample from the subject/peak area of Internal Standard). Free
lyso-substance concentrations were evaluated using an Internal
Standard methodA linear (y=ax+b) regression model using the
weighting factor 1/conc. will be used to calculate the
concentration of each analyte in every batch to be evaluated. The
concentrations were calculated by means of the following
formula:
concentration = peak area ratio - intercept ( b ) slope ( a )
##EQU00001##
[0337] Based thereon mean values, precision results (in terms of
CVs) and accuracies (formula shown below) will be calculated using
the program "Lotus 123".
accuracy ( % ) = calculated concentration expected concentration
100 ##EQU00002##
[0338] Appropriate statistical models are described in e.g. [0339]
Green, J. R., Statistical Treatment of Experimental Data (Elsevier,
New York, 1977), page 210 ff [0340] Lothar Sachs, Angewandte
Statistik--Anwendung statistischer Methoden (Springer, Berlin,
Heidelberg, New York, Tokyo 1984)
[0341] Software
[0342] Data acquisition, data processing, statistics and
calculations were performed using Analyst.RTM. software 1.4.2 or
higher (AB SCIEX, USA/Canada) as well as Lotus 1-2-3 97 or higher
(Lotus Corp, USA).
[0343] Handbooks [0344] Handbook Arbeiten mit SmartSuite 97 (Lotus
Development Corp., 1997) [0345] Documentation of software used
Documentation of Analyst.RTM. Software (AB SCIEX, USA/Canada):
[0346] Operator's Manual & Operator's Manual Addendum "New
Functionality in Analyst 1.2" and Online Help System Analyst 1.4
(or higher)
[0347] The protocols described 1 above were used to generate
HPLC-mass spectrometric chromatograms of 150 plasma samples derived
from the 150 subjects described in more details in Example 2.
Exemplary HPLC-mass spectrometric chromatograms displaying peak
intensity of free lyso-Gb1-sulfatide and IS of a healthy control
person and of a patient diagnosed as MDL-positive are depicted in
FIG. 1A and FIG. 1B.
[0348] More particularly, FIG. 1A shows HPLC-mass spectrometric
chromatograms displaying peak intensity in cps of free
lyso-Gb1-sulfatide (upper panel) and IS (lower panel) of a sample
from a healthy subject as a function over the retention time in
minutes. FIG. 1B shows HPLC-mass spectrometric chromatograms
displaying peak intensity in cps of free lyso-Gb1-sulfatide (upper
panel) and IS (lower panel) of a sample from an MLD patient as a
function over the retention time in minutes.
[0349] The retention time of a substance as used herein, preferably
is depicted on the x-axis and is the elapsed time between the time
of injection of a solute, e.g. a biomarker according to the present
invention and/or an internal standard, and the time of elution of
the peak maximum of said solute. A person skilled in the art will
acknowledge that the retention time of a substance according to the
herein described methods is a unique characteristic of said solute
and can be used for identification purposes. Internal Standard
working solution comprising lyso-Gb2 as an internal standard was
added to the sample as described above. It is important to
understand that by said addition of IS to the sample, i.e. spiking
of the sample, to be subjected to the method according to the
present invention, the concentration of IS in the sample is known
and by determining the area under the peak, i.e. the peak area, of
the internal standard in said HPLC-mass spectrometric chromatogram
the relation between a peak area and a concentration of a
substance, e.g. of IS and/or a biomarker thus can be calculated.
More precisely, a person skilled in the art will acknowledge that a
peak area of a substance depicted in an HPLC-mass spectrometric
chromatogram, such as the HPLC-mass spectrometric chromatogram
depicted in FIG. 1A or FIG. 1B represents a measure for an amount
of said substance subjected to an HPLC-mass spectrometric analysis.
Moreover, a person skilled in the art will be able to calculate the
amount of the substance in a sample from a subject subjected to an
HPLC-mass spectrometric analysis, e.g. the amount of free
lyso-Gb1-sulfatide in a sample subjected to the method of the
present invention, using a ratio of the peak area of free
lyso-Gb1-sulfatide, the amount of which is to be determined by said
method and the peak area of IS, e.g. free lyso-Gb2; as well as
calibration curves generated with said method and said free
lyso-Gb1-sulfatide and/or IS. Accordingly, this allows subsequently
for determining a level of free lyso-Gb1-sulfatide.
[0350] With regard to free lyso-Gb1-sulfatide<LLOQ (limit of
determination) has been replaced by 0.025, which refers to half of
the limit of detection.
Example 2: Genetic Testing and Classification of Study
Participants
[0351] After consenting of patients to participation in the study,
patients were subjected to a genetic testing for mutations of the
gene coding for ARSA. Accordingly, 5 to 10 ml of EDTA blood were
sequenced according to Seeman et al. (Seeman et al., 1995; Seeman,
N. C. (1995) J. Am. Chem. Soc. 117, 1194-1200; N. C. Seeman,
Biochemistry 34, 673-682 (1995); N. C. Seeman, Structural Domains
of DNA Mesojunctions, Biochemistry 34, 920-929 (1995); N. C.
Seeman, The Chemical Intelligencer 1(3), 38-47 (1995); N. C.
Seeman, Journal of the Chemical Society, Chemical Communications,
2249-2250 (1995)). Were appropriate other genes beside the gene
coding for ARSA were sequenced in addition, particularly in
controls. Said genetic testing was controlled using test samples of
age and sex matched control patients.
[0352] Plasma samples from a first cohort of 93 subjects were
analyzed.
[0353] The level of free lyso-Gb1-sulfatide in samples of said 93
subjects was determined according to the method described in
Example 1. Table 1 shows the median and minimum and maximum levels
of free lyso-Gb1-sulfatide in said samples of said 93 subjects. As
is evident from said Table 1, MLD patients could be clearly
distinguished from subjects suffering from other LSDs. Such other
LSDs included Fabry disease, Gaucher's disease and Nieman-Pick
disease type C.
TABLE-US-00008 TABLE 1 MLD other LSD's p n 25 68 median (IQR) 0.35
(0.19-0.62) 0.05 (0.05-0.05) <0.001 min-max 0.05-1.49
0.05-0.05
[0354] Plasma samples from a second cohort of 150 subjects were
analyzed, whereby such second cohort comprised the first
cohort.
[0355] The level of free lyso-Gb1-sulfatide in samples of said 150
subjects was determined according to the method described in
Example 1. In connection with said second cohort of 150 patients
183 samples have been analysed. That total group comprises 25 MLD
patients, 3 carriers, 6 healthy controls and 116 patients with
other lysosomal storage disorders: 5 Gaucher patients (27 samples),
20 Fabry unclear patients (20 samples), 11 male Fabry patients (15
samples), 9 Female Fabry patients (11 samples), 7 NPC1 patients (9
samples), 6 NPC1 carriers (9 samples), 5 NPA/B patients (5
samples), 6 Krabbe disease patients (6 samples), 8 male Hunter
disease patients (8 samples), 2 female Hunter disease patients (2
samples), 3 GM1 gangliosidosis patients (3 samples), 3 San Filippo
B patients (MPS3b) (3 samples), 9 MPS6 patients (9 samples), 1 MPS6
carrier (1 sample), 1 Tay-Sachs disease patient (1 sample), 4 MPS1
(4 samples), 3 San Filippo A patients (MPS3a) (3 samples), 12 MPS3a
patients (Morquio) (12 samples) and 1 MPS3a carrier (Morquio) (1
sample). Upon stratifying these patients into healthy controls
("healthy controls"), patients suffering from MLD ("MLD"), patients
being MLD carrier ("MLD carrier"), patients suffering from other
lysosomal storage diseases ("other LSD") results in a cohort
composition summarized in Table 2.
TABLE-US-00009 TABLE 2 frequency percentage Healthy controls 6 4.0
MLD 25 16.7 MLD carrier 3 2.0 Other LSD 116 77.3 Total 150
100.0
[0356] The results from the analysis of the second cohort are
depicted in FIG. 2.
[0357] FIG. 2 is a boxplot indicating levels of free lyso-Gb1
sulfatide; the y-axis indicates the absolute levels of free
lyso-Gb1-sulfatide in ng/ml determined in plasma of patients by the
method according to the present invention, wherein the x-axis
depicts groups of patients, which have been grouped as described.
The boxplot represents the 25th and 75th percentile of each group
of patients by the bottom and top of the box, respectively; the
band near the middle of the box represents the 50th percentile
(i.e. the median) of each group; The whiskers represent one
standard deviation above and below the mean of the data; Any data
not included between the whiskers is shown as an outlier with a
small circle
Example 3: Diagnosis of MLD Using Free Lyso-Gb1-Sulfatide as a
Biomarker
[0358] Plasma samples from the first cohort of patients defined in
Example 1 were subject to the methods and analysis described in
Example 1. The results from said analysis are depicted in FIG.
2
[0359] FIG. 2 is a boxplot indicating levels of free
lyso-Gb1-sulfatide in patients diagnosed either as MLD positive or
as suffering from other LSDs. The y-axis demonstrates the levels of
free lyso-Gb1-sulfatide in ng/ml determined in plasma of patients
by the method of Example 1, wherein the x-axis depicts groups of
patients, which have been grouped as described in Example 2. The
boxplot represents the 25.sup.th and 75.sup.th percentile of each
group of patients by the bottom and top of the box, respectively;
the band near the middle of the box represents the 50.sup.th
percentile (i.e. the median) of each group; the whiskers represent
one standard deviation above and below the mean of the data. Any
data not included between the whiskers is shown as an outlier with
a small circle or star.
[0360] Plasma samples from the second cohort of patients defined in
Example 1 were subject to the methods and analysis described in
Example 1. The results from said analysis are depicted in FIG.
3
[0361] FIG. 3 is a boxplot indicating levels of free lyso-Gb1
sulfatide; The y-axis indicates the absolute levels of free
lyso-Gb1-sulfatide in ng/ml determined in plasma of patients by the
method of Example 1, wherein the x-axis depicts groups of patients,
which have been grouped as described (healthy controls, patients
suffering from MLD, patients being MLD carrier and patients
suffering from LSD different from MLD). The boxplot represents the
25th and 75th percentile of each group of patients by the bottom
and top of the box, respectively; the band near the middle of the
box represents the 50th percentile (i.e. the median) of each group.
The whiskers represent one standard deviation above and below the
mean of the data. Any data not included between the whiskers is
shown as an outlier with a small circle.
[0362] For the second cohort of patients the activity of
arylsulfatase A was determined in plasma using standard
fluorometric procedures. The results from said analysis are
depicted in FIG. 4
[0363] FIG. 4 is a boxplot indicating the arylsulfatase enzyme
activity in the groups defined above. The y-axis demonstrates the
arylsulfatase enzyme given as U/mg protein, wherein the x-axis
depicts groups of patients, which have been grouped as described
above. The boxplot represents the 25th and 75th percentile of each
group of patients by the bottom and top of the box, respectively;
the band near the middle of the box represents the 50th percentile
(i.e. the median) of each group; the whiskers represent one
standard deviation above and below the mean of the data; any data
not included between the whiskers is shown as an outlier with a
small circle or star.
[0364] For comparing the diagnostic value of the different
biomarkers, namely free lyso-Gb1-sulfatide and enzymatic activity
of arylsulfatase A, and for the calculation of correlations between
the two biomarkers the data obtained by the method described above
was first aggregated by using the first measured value of every
marker for MLD patient.
[0365] Paired sample statistical techniques were used for the
comparison of two biomarkers. The method exploits the mathematical
equivalence of the AUC to the Mann-Whitney U-statistic (Delong E.
R., Delong D. M., Clarke-Pearson D. L., 1988, Biometrics, 44,
837-45.).
[0366] The accuracy of levels of the different biomarkers (free
lyso-Gb1-sulfatide) obtained by the method described in Example 1
above was evaluated to discriminate patients with MLD from patients
without having MLD using Receiver Operating Characteristic (ROC)
curve analysis (Metz C. E., 1978, Semin Nucl Med, 8, 283-98; Zweig
M. H., Campbell G., 1993, Clin Chem, 39, 561-77).
[0367] The ROC curves were calculated using PASW Statistics 18,
Release Version 18.0.2 (.COPYRGT. SPSS, Inc., 2009, Chicago, Ill.,
www.spss.com). The comparisons of ROC curves and the linear mixed
models were done using SAS software, Version 9.2 of the SAS System
for Windows. (.COPYRGT. 2008 SAS Institute Inc., Cary, N.C.,
USA).
[0368] The respective ROC curve is shown in FIG. 5.
[0369] FIG. 5 is a graph showing receiver operating characteristics
(ROC) curves of free lyso-Gb1--sulfatide and arylsulfatase A. The
x-axis represents "1-specificity" and the y-axis represents the
sensitivity. Free lyso-Gb1-sulfatide demonstrates a 98% sensitivity
and 99.1% specificity, wherein arylsulfatase A has at best a
sensitivity of 92.1% and 94% specificity, respectively.
[0370] The results are also summarized in Table 3.
TABLE-US-00010 TABLE 3 Enzymatic activity of Free lyso-Gb1 ARSA
[U/mg protein] sulfatide [ng/ml] (n = 150/incl. 25 mld) (n =
150/incl. 25 mld) Cut point <56 >0.011 Sensitivity 92.1%
98.0% Specificity 94.0% 99.1% AUC and 95% CI 0.992 (0.957-1.000)
0.997 (0.992-1.000) in ROC Analysis
Example 4: Incorrect Diagnosis of a Patient as MLD Positive Based
on Enzymatic Activity of Arylsulfatase A
[0371] Two patients demonstrated a pathological reduced enzyme
activity for aylsulfatse; however the lyso-Gb1-sulfatide was
normal; one patient was homozygous for the mutation p.N352S, the
second one was compound heterozygous.
[0372] In HGMD database (Human Gene Mutation Database at the
Institute of Medical Genetics in Cardiff) this mutation p.N352S is
published in 3 articles (Gieselmann 1989, Proc. Natal. Acad. Sci
USA; 86(23): 9436-40; Baronica 2011, Coll Antropol. 2011 January;
35 Suppl 1:11-6; and Rickettes 1996, Am J Med Genet. 67(4):
387-92). They associate p.N352S with ASA pseudodeficiency (in vitro
and in vivo studies show that this allele has a normal stability
and activity, but predicted to be less stable and less active).
Gieselmann 1989 shows as well that ARSA activity in tissues from
compound heterozygous individuals is lower than homozygous p.N352S
individuals. This is a disease-associated polymorphism in HGMD and,
most importantly, in dbSNP (rs2071421) the minor allele frequency
for this SNP is 20% in the general population and varies between
13-39% in the several hapmap populations. Finally, PolyPhen
prediction is "benign" (probability of being a mutation is
1.2%).
[0373] The features of the present invention disclosed in the
specification, the claims, the sequence listing and/or the drawings
may both separately and in any combination thereof be material for
realizing the invention in various forms thereof.
* * * * *
References