U.S. patent application number 16/083178 was filed with the patent office on 2019-03-21 for biomarkers of traumatic brain injury.
This patent application is currently assigned to The University of Birmingham. The applicant listed for this patent is The University of Birmingham. Invention is credited to Antonio Belli, Valentina Di Pietro.
Application Number | 20190085395 16/083178 |
Document ID | / |
Family ID | 55859162 |
Filed Date | 2019-03-21 |
United States Patent
Application |
20190085395 |
Kind Code |
A1 |
Belli; Antonio ; et
al. |
March 21, 2019 |
BIOMARKERS OF TRAUMATIC BRAIN INJURY
Abstract
Provided is a method of diagnosing and/or monitoring traumatic
brain injury (TBI) in a subject. The method comprises determining a
level of at least one miRNA in a fluid sample from the subject. The
miRNA may be selected from mat-425-5p, miR-502, milt-21 and
miR-335. The method may involve determining whether a subject is
suffering from mild-TBI or moderate-to-severe TBI. Also provided is
a sensor element, a detection system, composition and a kit for
diagnosing and/or monitoring TBI, and a method of determining an
appropriate treatment for a subject with a suspected TBI.
Inventors: |
Belli; Antonio; (East Wellow
Hampshire, GB) ; Di Pietro; Valentina; (West
Midlands, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Birmingham |
Birmingham |
|
GB |
|
|
Assignee: |
The University of
Birmingham
Birmingham
GB
|
Family ID: |
55859162 |
Appl. No.: |
16/083178 |
Filed: |
January 30, 2017 |
PCT Filed: |
January 30, 2017 |
PCT NO: |
PCT/GB2017/050231 |
371 Date: |
September 7, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/118 20130101;
C12Q 2600/178 20130101; C12Q 2600/106 20130101; C12Q 2600/158
20130101; A61P 9/00 20180101; C12Q 1/6883 20130101; A61P 25/00
20180101; A61P 25/20 20180101 |
International
Class: |
C12Q 1/6883 20060101
C12Q001/6883 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2016 |
GB |
1603967.9 |
Claims
1. A method of diagnosing and/or monitoring traumatic brain injury
(TBI) in a subject, the method comprising determining a level of at
least one miRNA in a fluid sample from the subject.
2. The method of claim 1, wherein the at least one miRNA is
selected from the group consisting of: miR-505, miR-203,
miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502,
miR-21, miR-let-7g, miR-335, miR-126*, miR-193a-5p, miR-144*,
miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625*,
miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p miR-142-3p, miR-148a-3p,
miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p,
miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p,
miR-424-5p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p;
miR-324-5p; miR-652-3p; miR-10a, miR-132, miR-223, miR-143,
miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a,
miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a,
miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any
combination thereof.
3. The method of claim 1 or claim 2, wherein the at least one miRNA
is selected from the group consisting of miR-505, miR-203,
miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502,
miR-21, miR-let-7g, miR-335, hsa-miR-126*, miR-193a-5p, miR-144*,
miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625*, and
miR-671-3p, or any combination thereof.
4. The method of any one of claims 1 to 3, wherein the at least one
miRNA is selected from the group consisting of miR-505, miR-203,
miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502,
miR-21, miR-let-7g and miR-335, or any combination thereof.
5. The method of claim 1 or claim 2, wherein the at least one miRNA
is selected from the group consisting of let-7c-5p, let-7i-5p,
miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p,
miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p,
miR-29a-3p, miR-29c-3p, and miR-424-5p; miR-30a-5p; miR-107;
miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, or any
combination thereof.
6. The method of claim 1 or claim 2, wherein the at least one miRNA
is selected from the group consisting of miR-10a, miR-132, miR-223,
miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95,
miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345,
miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and
miR-629, or any combination thereof.
7. The method of any one of claims 1 to 4, wherein the at least one
miRNA is selected from the group consisting of: miR-425-5p;
miR-502; miR-21; and miR-335, or any combination thereof.
8. The method of claim 7, wherein the TBI is mild TBI (mTBI) and
the miRNA is selected from the group consisting of: miR-425-5p and
miR-502.
9. The method of claim 8, wherein the subject is diagnosed as
having mTBI if the level of miR-425-5p and/or miR-502 is determined
to be below a predetermined threshold or decreased relative to a
reference.
10. The method of claim 7, wherein the TBI is severe TBI (sTBI) and
the miRNA is selected from the group consisting of: miR-21 and
miR-335.
11. The method of claim 10, wherein the subject is diagnosed as
having sTBI if the level of miR-21 and miR-335 is determined to be
above a predetermined threshold or increased relative to a
reference.
12. The method of claim 7, wherein the method comprises determining
the level of: (i) a first miRNA selected from miR-425-5p and
miR-502; and (ii) a second miRNA selected from miR-21 and
miR-335.
13. The method of claim 12, wherein the subject is diagnosed as
having a TBI of any severity if the level of miR-425-5p or miR-502
is determined to be below a predetermined threshold, or is
decreased relative to a reference, and the level of miR-21 or
miR-335 is determined to be above a predetermined threshold, or is
increased relative to a reference.
14. The method of any preceding claim, wherein the method is for
diagnosing TBI and the level of the at least one miRNA is
determined in a fluid sample obtained from the subject no more than
48 hours after injury.
15. The method of any preceding claim, wherein the fluid sample
comprises saliva, blood, plasma or serum.
16. The method of claim 15, wherein the fluid sample is saliva.
17. A sensor element for a detection system for diagnosing and/or
monitoring TBI, the sensor element comprising a substrate
functionalized with a probe specific for a target miRNA.
18. The sensor element of claim 17, wherein the probe comprises a
nucleic acid.
19. The sensor element of claim 18, wherein the nucleic acid
comprises a sequence which is at least 80% identical to a sequence
which is the complement of the sequence of the target miRNA.
20. The sensor element of any one of claims 17 to 19, wherein the
substrate is formed from metal, plastic, glass, silica, silicon,
graphite or graphene, or any combination thereof.
21. A detection system for diagnosing and/or monitoring TBI,
comprising: a sensor element according to any one of claims 17 to
20; and a detection device that is capable of detecting the binding
of a target miRNA to the probe.
22. A kit for use in a method of diagnosing and/or monitoring
traumatic brain injury (TBI) in a fluid sample from a subject, the
kit comprising at least one probe specific for a target miRNA.
23. A method for determining whether it is appropriate to
administer to a subject a therapy for alleviating TBI, the method
comprising: determining a level of at least one miRNA in a fluid
sample from the subject; and determining whether or not it is
appropriate to administer a therapy for alleviating TBI, based on
the level of the at least one miRNA.
24. A method of determining an appropriate treatment of a subject
suspected of suffering from a TBI, the method comprising
identifying whether or not the subject has a TBI by determining a
level of at least one miRNA in a fluid sample from the subject.
25. The method of claim 24, wherein if the subject is identified as
having a TBI, an appropriate treatment is determined to be at least
one of: further evaluating the subject; removing the subject from
activity; admitting the subject to a hospital or clinic; and
administering a therapy for alleviating TBI to the subject.
26. A therapy for alleviating TBI for use in a method of treating a
subject in need thereof, wherein said subject is identified as
having a TBI by determining a level of at least one miRNA in a
fluid sample from the subject.
27. The method of any one of claims 24 to 26, wherein the therapy
for alleviating TBI is selected from at least one of:
neuroprotective drugs, a sedative; and avoidance of the use of
hypertensive resuscitation.
28. A composition for use in a method of diagnosing and/or
monitoring traumatic brain injury (TBI) in a subject, the
composition comprising a probe specific for a target miRNA.
29. The composition of claim 28, comprising a plurality of
probes.
30. The sensor element, detection system, kit, method or
composition according to claims 17 to 29, wherein the miRNA is
selected from the group consisting of: miR-505, miR-203,
miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502,
miR-21, miR-let-7g, miR-335, miR-126*, miR-193a-5p, miR-144*,
miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625*,
miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p,
miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p,
miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p,
miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p;
miR-652-3p, miR-424-5p, miR-10a, miR-132, miR-223, miR-143,
miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a,
miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a,
miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any
combination thereof.
31. The sensor element, detection system, kit, method or
composition according to claim 30, wherein the miRNA is selected
from the group consisting of miR-425-5p; miR-502; miR-21; and
miR-335, or any combination thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions, kits, systems
and methods for diagnosing and/or monitoring traumatic brain injury
(TBI). More particularly, the present invention relates to the
diagnosis and monitoring of TBI using miRNA biomarkers.
BACKGROUND TO THE INVENTION
[0002] Traumatic brain injury (TBI) is the leading cause of death
and disability under the age of 45 years in Western countries. Its
healthcare burden and social costs are expected to continue to rise
and, by 2020, the World Health Organization projects TBI to become
the third leading cause of disability worldwide.
[0003] Despite many studies, no reliable biomarkers have been found
to assess the severity of TBI and predict recovery. This is
especially true for mild TBI (mTBI), which remains currently
difficult to assess in clinical practice. Although TBI patients are
initially assessed by the Glasgow Coma Score (GCS) and neuroimaging
techniques, which require costly equipment, the current diagnostic
tools are lacking in the ability to precisely define and quantify
the actual severity of the brain injury, thus leading to an easy
detection of severe but not of mTBI, which represents the vast
majority of cases (75-90%).
[0004] The correct diagnosis of mTBI is particularly important in
patients, such as athletes, soldiers and children, who are at
greater risk of repetitive mTBI and a catastrophic form of brain
injury known as second impact syndrome (SIS) where the synergistic
effects of repeated TBI result in profound damage and even death.
Early diagnosis and evaluation of the severity of TBI thus becomes
crucial for patients' wellbeing and ultimately saving their
life.
[0005] The quest for TBI biomarkers has received significant
impetus by the increased profile of sport concussion in the media.
In the last few years many studies have focused on biomarkers that
can support clinical decision making pitch-side or in a sports
clinic. However, protein biomarkers reported in the literature lack
specificity or sensitivity, or are not detectable for some time
after injury. This may be due to the fact that following
concussion, which is a form of TBI, brain-derived compounds are
only released in very small amounts and the blood-brain barrier
remains mostly closed.
[0006] MicroRNAs (miRNAs) are an abundant class of highly
conserved, non-coding RNA molecules of approximately 22 nucleotides
in length that induce mRNA degradation, translational repression or
both via pairing with partially complementary sites in the 3' UTR
of target genes. The human genome encodes over 2,000 miRNAs, which
may target about 60% of all genes. However, despite the abundance
of miRNAs, their biomolecular functions and involvement in
pathology remain to be fully elucidated. They play a central role
in many biological processes including cell cycle, cell metabolism,
apoptosis and immune responses, and are attracting increasing
interest in clinical research as potential biomarkers for the
detection, identification and classification of cancers and other
disease states including neurodegenerative diseases.
[0007] The present invention was devised with these issues in
mind.
SUMMARY OF THE INVENTION
[0008] The present invention provides a method of diagnosing or
monitoring traumatic brain injury (TBI) in a subject.
[0009] According to a first aspect of the invention, there is
provided a method for diagnosing and/or monitoring traumatic brain
injury (TBI) in a subject, the method comprising detecting the
presence of and/or determining a level of at least one miRNA in a
sample from the subject.
[0010] The at least one miRNA (also referred to herein as "miR")
may be selected from the group consisting of miR-505, miR-203,
miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502,
miR-21, miR-let-7g, miR-335, miR-126*, miR-193a-5p, miR-144*,
miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625*,
miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p miR-142-3p, miR-148a-3p,
miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p,
miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p,
miR-424-5p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p;
miR-324-5p; miR-652-3p; miR-10a, miR-132, miR-223, miR-143,
miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a,
miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a,
miR-579, miR-642, miR-99a, miR-520D-3p and miR-629. These miRNAs
may be referred to herein as miRNAs of interest or target
miRNAs.
[0011] In some embodiments, the at least one miRNA is selected from
the group consisting of miR-505, miR-203, miR-654-3p, miR-655,
miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g,
miR-335, hsa-miR-126*, miR-193a-5p, miR-144*, miR-190, miR-194,
miR-365, miR-590-3p, miR-624, miR-625*, and miR-671-3p. These
microRNAs have been found to be biomarkers expressed in all TBI
patients (mild or severe).
[0012] In some embodiments, the at least one miRNA is selected from
the group consisting of miR-505, miR-203, miR-654-3p, miR-655,
miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g and
miR-335.
[0013] In some embodiments, the at least one miRNA is selected from
the group consisting of let-7c-5p, let-7i-5p, miR-142-3p,
miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p,
miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p,
miR-29c-3p, and miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p;
miR-199b-5p; miR-324-5p; miR-652-3p.
[0014] In some embodiments, the at least one miRNA is selected from
the group consisting of miR-10a, miR-132, miR-223, miR-143,
miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a,
miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361,
miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629.
[0015] For the avoidance of doubt, it will be understood that "the
at least one miRNA is selected from a group of miRNAs", as used
herein, means that the method in question, whether carried out for
a diagnostic, prognostic, or therapeutic purpose, can be carried
out with any one of the listed miRNAs or with any plurality of the
listed miRNAs (e.g., two, three, four, or more of the listed
miRNAs). It follows that any one or more of the listed miRNAs may
be explicitly excluded. For example, where the at least one miRNA
is selected from the group consisting of miR-505, miR-203,
miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502,
miR-21, miR-let-7g and miR-335, the method may include detecting
and/or assessing the level of any combination of miR-505, miR-203,
miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502,
miR-21, and miR-let-7g to the exclusion of miR-335.
[0016] According to an aspect of the invention, there is provided a
method of diagnosing and/or monitoring traumatic brain injury (TBI)
in a subject, the method comprising determining a level of at least
one miRNA in a sample from the subject, wherein the miRNA is
selected from the group consisting of: miR-425-5p; miR-502; miR-21;
and miR-335.
[0017] Traumatic brain injury occurs when an external force
traumatically injures the brain. There are different systems for
classifying TBI based on, for example, severity, type of injury and
prognosis. The most commonly used system for classifying TBI is the
Glasgow Coma Scale (GCS), which grades a person's level of
consciousness on a scale of 3-15 based on verbal, motor, and
eye-opening reactions to stimuli. In general, a TBI with a GCS
score of 13 or above is defined as mild, 9-12 as moderate and 8 or
below as severe. Another system, the Mayo Classification System,
has three main classifications including definite moderate-severe
TBI, probable mild TBI, and possible TBI. Multiple criteria are
used in each diagnosis including loss of consciousness,
post-traumatic amnesia, skull fracture, and evidence of
neuroradiological abnormalities including subdural haematoma,
cerebral contusion, and hemorrhagic contusion. The classification
of TBI using the GCS or Mayo systems will be known to those skilled
in the art.
[0018] As used herein, references to "mild", "moderate" and
"severe" TBI are made in accordance with the GCS. References herein
to "moderate-to-severe" TBI encompass both moderate and severe TBI
in accordance with the GCS.
[0019] The diagnosis and/or monitoring of TBI using the biomarkers
of the present invention is expected to support clinical decision
making and treatment regimens in a variety of contexts, including
the following situations: as part of the initial assessment by
paramedics to determine whether patients should be transported to a
facility with neurosurgical expertise, a major trauma centre or a
local trauma unit; in the emergency department of hospitals to
determine appropriate treatment, including the need for a CT brain
scan; pitch-side, to assist decision making as to the removal of a
player from play and assessment of the need to take a player to
hospital; in sports clinics, to confirm a concussive event and
enable decision making with regard to returning to play; in combat
situations, to determine the need to dispatch a rescue team and
evacuate a victim. Thus, subjects for whom the present invention
provides particular benefit include accident victims, sports
players and military personnel.
[0020] In any case, but perhaps particularly where the subject is
at greater risk for a TBI (e.g., where the subject is a
professional athlete or enlisted in the military), a sample may be
obtained from the subject at a time prior to any known or recent
trauma (e.g., near the beginning of a sporting career or prior to a
military deployment) and any miRNAs of interest can be assessed at
that time or later, when the subject has experienced a possible
TBI. Such samples may thereby provide an internal reference
standard.
[0021] In some embodiments, the subject is human.
[0022] The TBI may be mild TBI (mTBI), moderate TBI or severe TBI
(sTBI). In some embodiments, the TBI is moderate-to-severe TBI
(m-sTBI).
[0023] The level of the miRNA or of each miRNA in the sample may be
determined quantitatively or semi-quantitatively. By
"quantitatively", it will be understood that the absolute amount or
concentration of the miRNA or of each miRNA in the sample is
determined. The absolute amount of the miRNA or of each miRNA in
the sample can then be compared to a predetermined threshold (e.g.
a published literature value for expected normal levels), a known
level of the same or a reference miRNA in a control sample taken
from a healthy subject, or the amount of a reference miRNA in the
sample taken from the subject. In some embodiments, the subject is
diagnosed as having a TBI when the level of the miRNA is below the
predetermined threshold, or decreased relative to a reference or
control sample. In other embodiments, the subject is diagnosed as
having a TBI when the level of the miRNA is increased compared to
the predetermined threshold.
[0024] By "semi-quantitatively", it will be understood that the
level of the or each miRNA of interest is measured relative to a
reference.
[0025] The reference may be an invariant miRNA, i.e. a miRNA having
an expression level that remains substantially unchanged between
healthy subjects and those having a TBI. A subject may be diagnosed
as suffering from a TBI if the level of the miRNA or of each miRNA
of interest is increased or decreased relative to that of an
invariant miRNA. Suitable invariant miRNAs include miR-331,
miR-223*, miR-23a-3p and miR148b-3p. miR-23a-3p and miR148b-3p are
invariant in saliva only.
[0026] In some embodiments, the level of the miRNA or of each miRNA
in the sample obtained from the subject may be about 0.01 times to
about 100 times, about 0.05 times to about 50 times, about 0.1
times to about 10 times, about 0.5 times to about 5 times, about
1.0 to about 3 times, or about 1.5 times to about 2.0 times lower
or higher than the level in the control sample, the reference level
or the published value.
[0027] Where a device or method is employed to generate a value, we
may qualify that value with the term "about" in order to capture
the stated value and any variation of that value inherent to the
device or method employed. Where values or ranges of values are
specifically disclosed, "about" may mean plus-or-minus 10% of the
stated value or range. For example, about 10 minutes may mean 9-11
minutes.
[0028] The level of the miRNA or of each miRNA of interest can be
determined using methods known to those skilled in the art. In some
embodiments, determining the level of the miRNA or of each miRNA of
interest comprises amplifying the miRNA. In some embodiments, total
miRNA may be first isolated from the sample using standard
techniques, for example using the miRNeasy mini kit (Qiagen). The
amount of the miRNA of interest can then be determined. In some
embodiments, the level of the miRNA or of each miRNA of interest in
the sample is determined using PCR (polymerase chain reaction). For
example, quantitative PCR may be used for quantitative
determination of the level of the miRNA or of each miRNA of
interest. PCR may also be used for semi-quantitative determination,
by comparing the level of the miRNA or of each miRNA of interest in
the sample with that of a reference (e.g. an invariant miRNA).
[0029] Suitable techniques for miRNA detection and/or
quantification, which will be known to those skilled in the art,
include qPCR, miRNA assays, next-generation sequencing (NGS), and
multiplex miRNA profiling assays.
[0030] In some embodiments, the level of the miRNA or of each miRNA
of interest is determined using in-situ hybridization, for example
using a probe (e.g., a labelled probe) specific for the miRNA.
[0031] The level of miRNA may be determined in a sample which was
obtained from the subject immediately after injury (i.e. less than
1 hour after injury), and/or in a sample obtained at one or more
time points a few hours or days after injury. Thus, changes in the
miRNA level can be detected over time to enable monitoring of a
TBI. In the event miRNA levels change over time, the methods
described herein for monitoring TBI can be expanded to include
maintaining or adjusting the subject's treatment regimen
accordingly.
[0032] Depending on the specific miRNA and the type of TBI, the
level of miRNA in the subject may change significantly over time.
In some embodiments, it may therefore be advantageous to measure
the miRNA relatively soon after injury to enable an accurate
diagnosis. In some embodiments, the level of miRNA is determined in
a sample obtained from the subject no more than 72 hours, no more
than 48 hours, no more than 36 hours, no more than 24 hours, no
more than 12 hours, no more than 6 hours, no more than 4 hours, no
more than 2 hours or no more than 1 hour after injury.
[0033] The level of some miRNAs is substantially stable over time,
thus allowing a diagnosis to be made a few hours, days or even
weeks after injury. In some embodiments, the level of miRNA is
determined in a sample obtained from the subject up to 20, 18, 15,
12, 10, 8, 5 or 2 days from injury.
[0034] In some embodiments, the level of miRNA is determined in a
sample obtained from the subject immediately after injury (e.g. at
T=0h), at 4-12 hours after injury, at 48-72 hours after injury, or
at 15 days after injury.
[0035] In some embodiments, the TBI is mild TBI (mTBI) or
moderate-to-severe TBI (m-sTBI) and the at least one miRNA is
selected from the group consisting of miR-505, miR-203, miR-654-3p,
miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21,
miR-let-7g, miR-335, hsa-miR-126*, miR-193a-5p, miR-144*, miR-190,
miR-194, miR-365, miR-590-3p, miR-624, miR-625*, and
miR-671-3p.
[0036] In some embodiments, the TBI is mild TBI (mTBI) and the
miRNA is selected from the group consisting of miR-425-5p and
miR-502. The subject may be diagnosed as having mTBI if the level
of miR-425-5p and/or miR-502 is determined to be below a
predetermined threshold, or is decreased relative to a
reference.
[0037] In some embodiments, a level of miR-425-5p and/or miR-502
which is below a predetermined threshold, or decreased relative to
a reference, is diagnostic of mTBI when the level is determined in
a sample obtained less than 48 hours after injury.
[0038] In some embodiments, the TBI is moderate-to-severe TBI
(m-sTBI) and the miRNA is selected from the group consisting of
miR-21 and miR-335. The subject may be diagnosed as having
moderate-to-severe TBI if the level of miR-21 and/or miR-335 is
determined to be above a predetermined threshold, or increased
relative to a reference.
[0039] In some embodiments, a level of miR-21 and/or miR-335 which
is above a predetermined threshold, or increased relative to a
reference, is diagnostic of moderate-to-severe TBI when the level
is determined in a sample obtained up to 15 days after injury.
[0040] In some embodiments, the TBI is moderate-to-severe TBI
(m-sTBI) and the at least one miRNA is selected from the group
consisting of miR-10a, miR-132, miR-223, miR-143, miR-148b,
miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152,
miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a,
miR-579, miR-642, miR-99a, miR-520D-3p and miR-629.
[0041] The subject may be diagnosed as having m-sTBI if the level
of miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-618,
miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326,
miR-345, miR-361, miR-422a, miR-579, miR-642 and/or miR-99a is
determined to be above a predetermined threshold, or is increased
relative to a reference.
[0042] The subject may be diagnosed as having m-sTBI if the level
of miR-192, miR-429, miR-520D-3p and/or miR-629 is determined to be
below a predetermined threshold, or is decreased relative to a
reference.
[0043] The miRNAs may be used individually to diagnose TBI. For
example, in sport concussion, miR-502 or miR-425-5p could be used
to confirm that a traumatic brain injury has occurred.
[0044] Thus, a further aspect of the present invention is a method
of determining the severity of TBI, and the steps of this method
can be repeated to monitor the subject over time. It will be
appreciated that a positive result for a single miRNA (e.g. the
level of a single miRNA is determined to be above/below a
predetermined threshold, or is increased/decreased relative to a
reference) is sufficient to determine the severity of TBI. For
example, if the level of miR-425-5p is below a predetermined
threshold, or decreased relative to a reference, the severity of
the TBI is determined to be mild (mTBI). However, it may be
convenient to combine different miRNAs (e.g. in a test panel) to
facilitate the assessment of TBI severity.
[0045] In some embodiments, the method comprises determining a
level of a plurality (e.g., two or more) miRNAs in the sample. In
some embodiments the two or more miRNAs are selected from the group
consisting of: miR-425-5p; miR-502; miR-21; and miR-335.
[0046] In some embodiments, the method comprises determining the
level of:
(i) a first miRNA selected from miR-425-5p and miR-502; and (ii) a
second miRNA selected from miR-21 and miR-335.
[0047] A subject may be diagnosed as having a TBI if the level of
miR-425-5p or miR-502 is determined to be below a predetermined
threshold, or is decreased relative to a reference, or the level of
miR-21 or miR-335 is determined to be above a predetermined
threshold, or increased relative to a reference.
[0048] In some embodiments, the TBI is mild TBI (mTBI) and the at
least one miRNA is selected from the group consisting of let-7c-5p,
let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p,
miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miRmmiR-29a-3p,
miR-29c-3p, miR-424-5p, miR-30a-5p, miR-107, miR-135b-5p,
miR-199b-5p, miR-324-5p, and miR-652-3p, or a combination thereof.
The subject may be diagnosed as having mTBI if there is a fold
change of at least 0.5, at least 1.0, at least 1.5, at least 2.0,
at least 2.5, at least 3.0, at least 3.5 or at least 4.0 in level
of the microRNA compared to a reference. In some embodiments, the
subject is diagnosed as having mTBI if the level(s) of the
microRNA(s) is/are increased compared to a reference.
[0049] The sequences and accession numbers for miRNAs described
herein are provided in Table 1:
TABLE-US-00001 TABLE 1 miRBase miRNA sequence Accession no.
hsa-miR-21 uagcuuaucagacugauguuga (SEQ ID No. 1) MIMAT0000076
hsa-miR-425-5p aaugacacgaucacucccguuga (SEQ ID No. 2) MIMAT0003393
hsa-miR-502-5p (also auccuugcuaucugggugcua (SEQ ID No. 3)
MIMAT0002873 known as hsa-miR-502) hsa-miR-335-5p (also
ucaagagcaauaacgaaaaaugu (SEQ ID No. 4) MIMAT0000765 known as
hsa-miR-335) hsa-miR-301b-3p (also cagugcaaugauauugucaaagc (SEQ ID
No. 5) MIMAT0004958 known as hsa-miR-301b) hsa-miR-184
uggacggagaacugauaagggu (SEQ ID No. 6) MIMAT0000454 hsa-miR-505-3p
(also cgucaacacuugcugguuuccu (SEQ ID No. 7) MIMAT0002876 known as
hsa-miR-505) hsa-miR-203a-3p (also gugaaauguuuaggaccacuag (SEQ ID
No. 8) MIMAT0000264 known as hsa-miR-203a, hsa-miR-203)
hsa-miR-654-3p uaugucugcugaccaucaccuu (SEQ ID No. 9) MIMAT0004814
hsa-miR-655-3p (also auaauacaugguuaaccucuuu (SEQ ID No. 10)
MIMAT0003331 known as hsa-miR-655) hsa-miR-331-3p (also
gccccugggccuauccuagaa (SEQ ID No. 11) MIMAT0000760 known as
hsa-miR-331) hsa-miR-223-5p (also cguguauuugacaagcugaguu (SEQ ID
No. 12) MIMAT0004570 known as hsa-miR-223*) hsa-miR-let-7g
ugagguaguaguuuguacaguu (SEQ ID No. 13) MIMAT0000414 hsa-miR-126*
(also known cauuauuacuuuugguacgcg (SEQ ID No. 14) MIMAT0000444 as
hsa-miR-126-5p) hsa-miR-193a-5p ugggucuuugcgggcgagauga (SEQ ID No.
15) MIMAT0004614 hsa-miR-144* (also known ggauaucaucauauacuguaag
(SEQ ID No. 16) MIMAT0004600 as hsa-miR-144-5p) hsa-miR-190 (also
known cuauauaucaaacauauuccu (SEQ ID No. 17) MIMAT0026482 as
hsa-miR-190a or hsa- miR-190a-3p) hsa-miR-194 (also known
uguaacagcaacuccaugugga (SEQ ID No. 18) MIMAT0000460 as
hsa-miR-194-5p) hsa-miR-365 (also known uaaugccccuaaaaauccuuau (SEQ
ID No. 19) MIMAT0000710 as hsa-miR-365a-3p) hsa-miR-590-3p (also
uaauuuuauguauaagcuagu (SEQ ID No. 20) MIMAT0004801 known as
hsa-miR-590) hsa-miR-624 uaguaccaguaccuuguguuca (SEQ ID No. 21)
MI0003638 hsa-miR-625* (also known gacuauagaacuuucccccuca (SEQ ID
No. 22) MIMAT0004808 as hsa-miR-625-3p) hsa-miR-671-3p
uccgguucucagggcuccacc (SEQ ID No. 23) MIMAT0004819 hsa-let-7c-5p
ugagguaguagguuguaugguu (SEQ ID No. 24) MIMAT0000064 hsa-let-7i-5p
(also ugagguaguaguuugugcuguu (SEQ ID No. 25) MIMAT0000415 known as
hsa-let-7i) hsa-miR-142-3p uguaguguuuccuacuuuaugga (SEQ ID No. 26)
MIMAT0000434 hsa-miR-148a-3p (also ucagugcacuacagaacuuugu (SEQ ID
No. 27) MIMAT0000243 known as hsa-miR-148a) hsa-miR-15b-5p (also
uagcagcacaucaugguuuaca (SEQ ID No. 28) MIMAT0000417 known as
hsa-miR-15b) hsa-miR-16-5p (also uagcagcacguaaauauuggcg (SEQ ID No.
29) MIMAT0000069 known as hsa-miR-16) hsa-miR-181a-5p (also
aacauucaacgcugucggugagu (SEQ ID No. 30) MIMAT0000256 known as
hsa-miR-181a) hsa-miR-20a-5p (also uaaagugcuuauagugcagguag (SEQ ID
No. 31) MIMAT0000075 known as hsa-miR-20; hsa-miR-20a)
hsa-miR-20b-5p (also caaagugcucauagugcagguag (SEQ ID No. 32)
MIMAT0001413 known as hsa-miR-20b) hsa-miR-221-3p (also
agcuacauugucugcuggguuuc (SEQ ID No. 33) MIMAT0000278 known as
hsa-miR-221) hsa-miR-24-3p (also uggcucaguucagcaggaacag (SEQ ID No.
34) MIMAT0000080 known as hsa-miR-24) hsa-miR-27b-3p (also
uucacaguggcuaaguucugc (SEQ ID No. 35) MIMAT0000419 known as
hsa-miR-27b) hsa-miR-29a-3p (also uagcaccaucugaaaucgguua (SEQ ID
No. 36) MIMAT0000086 known as hsa-miR-29a) hsa-miR-29c-3p (also
uagcaccauuugaaaucgguua (SEQ ID No. 37) MIMAT0000681 known as
hsa-miR-29c) hsa-miR-30a-5p uguaaacauccucgacuggaag (SEQ ID No. 38)
MIMAT0000087 (also known as hsa-miR- 30a) hsa-miR-107 (also known
agcagcauuguacagggcuauca (SEQ ID No. 39) MI0000114 as
hsa-miR-107-10) hsa-miR-135b-5p (also uauggcuuuucauuccuauguga (SEQ
ID No. 40) MIMAT0000758 known as hsa-miR-135b) hsa-miR-199b-5p
(also cccaguguuuagacuaucuguuc (SEQ ID No. 41) MIMAT0000263 known as
hsa-miR-199b) hsa-miR-324-5p cgcauccccuagggcauuggugu (SEQ ID No.
42) MIMAT0000761 hsa-miR-652-3p (also aauggcgccacuaggguugug (SEQ ID
No. 43) MIMAT0003322 known as hsa-miR-652) hsa-miR-424-5p (also
cagcagcaauucauguuuugaa (SEQ ID No. 44) MIMAT0001341 known as
hsa-miR-424) hsa-miR-10a (also known uacccuguagauccgaauuugug (SEQ
ID No. 45) MIMAT0000253 as hsa-miR-10-5p) hsa-miR-132 (also known
uaacagucuacagccauggucg (SEQ ID No. 46) MIMAT0000426 as
hsa-miR-132-3p) hsa-miR-223 (also known ugucaguuugucaaauacccca (SEQ
ID No. 47) MIMAT0000280 as hsa-miR-223-3p) hsa-miR-143 (also known
ugagaugaagcacuguagcuc (SEQ ID No. 48) MIMAT0000435 as
hsa-miR-143-3p) hsa-miR-148b (also known ucagugcaucacagaacuuugu
(SEQ ID No. 49) MIMAT0000759 as hsa-miR-148b-3p) hsa-miR-18a (also
known uaaggugcaucuagugcagauag (SEQ ID No. 50) MIMAT0000072 as
hsa-miR-18; hsa-miR- 18a-5p) hsa-miR-192 (also known
cugaccuaugaauugacagcc (SEQ ID No. 51) MIMAT0000222 as
hsa-miR-192-5p) hsa-miR-429 uaauacugucugguaaaaccgu (SEQ ID No. 52)
MIMAT0001536 hsa-miR-618 aaacucuacuuguccuucugagu (SEQ ID No. 53)
MIMAT0003287 hsa-miR-95 (also known ucaauaaaugucuguugaauu (SEQ ID
No. 54) MIMAT0026473 as hsa-miR-95-5p) hsa-miR-130a (also known
cagugcaauguuaaaagggcau (SEQ ID No. 55) MIMAT0000425 as
hsa-miR-130a-3p) hsa-miR-152 (also known agguucugugauacacuccgacu
(SEQ ID No. 56) MIMAT0026479 as hsa-miR-152-5p) hsa-miR-27b (also
known uucacaguggcuaaguucugc (SEQ ID No. 57) MIMAT0000419 as
hsa-miR-27b-3p) hsa-miR-301 (also known cagugcaauaguauugucaaagc
(SEQ ID No. 58) MIMAT0000688 as hsa-miR-301a-3p or hsa-miR-301a)
hsa-miR-326 ccucugggcccuuccuccag (SEQ ID No. 59) MIMAT0000756
hsa-miR-345 (also known gcugacuccuaguccagggcuc (SEQ ID No. 60)
MIMAT0000772 as hsa-miR-345-5p) hsa-miR-361 (also known
uuaucagaaucuccagggguac (SEQ ID No. 61) MIMAT0000703 as
hsa-miR-361-5p) hsa-miR-422a acuggacuuagggucagaaggc (SEQ ID No. 62)
MIMAT0001339 hsa-miR-579 (also known uucauuugguauaaaccgcgauu (SEQ
ID No. 63) MIMAT0003244 as hsa-miR-579-3p) hsa-miR-642 (also known
gucccucuccaaaugugucuug (SEQ ID No. 64) MIMAT0003312 as
hsa-miR-642a-5p; hsa-miR-642) hsa-miR-99a (also known
aacccguagauccgaucuugug (SEQ ID No. 65) MIMAT0000097 as
hsa-miR-99a-5p) hsa-miR-520D-3p (also aaagugcuucucuuuggugggu (SEQ
ID No. 66) MIMAT0002856 know as hsa-miR-520D) hsa-miR-629 (also
known uggguuuacguugggagaacu (SEQ ID No. 67) MIMAT0004810 as
hsa-miR-629-5p) hsa-miR-23a-3p (also aucacauugccagggauuucc (SEQ ID
No. 68) MIMAT0000078 known as hsa-miR-23a) hsa-miR-148b-3p (also
ucagugcaucacagaacuuugu (SEQ ID No. 69) MIMAT0000759 known as
hsa-miR-148b)
[0050] Conveniently the sample may be any appropriate fluid or
tissue sample obtained from the subject. For example, the
biological sample may comprise at least one of the group consisting
of: urine, saliva, whole blood, plasma, serum, sputum, semen,
faeces, a nasal swab, tears, a vaginal swab, a rectal swab, a
cervical smear, a tissue biopsy, and a urethral swab. In some
embodiments the sample is a fluid sample. Suitably, the sample is
one that can be readily obtained from the individual, such as
urine, saliva, blood and sputum. In some embodiments, the sample
comprises saliva, blood, plasma or serum. It will be appreciated
that in some embodiments the process of obtaining the sample does
not form part of the invention described herein.
[0051] In some embodiments, the sample comprises or is constituted
by serum. Not only does serum have practical advantages, but it is
also free of anticoagulants such as heparin, a potential inhibitor
of PCR reactions. Serum may also be less affected by haemolysis,
compared to plasma.
[0052] In some embodiments, the sample is saliva. Saliva can be
easily obtained from the patient (e.g. pitch-side, or in the
field), without specialist training or medical equipment.
[0053] miRNAs which have been found to be indicative of mTBI in
saliva include: hsa-let-7ca-5p, hsa-let-7i-5p, hsa-miR-1421-3p,
hsa-miR-148a-3p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-181a-5p,
hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-221-3p, hsa-miR-24-3p,
hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-340-5p,
hsa-miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b is
selected from the group consisting of -5p; miR-324-5p; and
miR-652-3p.
[0054] Levels of miRNAs may be used to track recovery of a subject
from injury. Thus, the present invention encompasses monitoring the
recovery of a subject from TBI, as an alternative or in addition to
the initial diagnosis.
[0055] In some embodiments, the method comprises monitoring TBI and
the level of the at least one miRNA is determined in a sample
obtained from the subject at least 2, at least 3, at least 5, at
least 7, at least 10 or at least 14 days after injury. In some
embodiments, the level of the at least one miRNA is determined in a
sample obtained from the subject 15 days after injury. In some
embodiments, the level of the at least one miRNA is determined in
at least two samples obtained at different time intervals after
injury, thus allowing recovery to be monitored. For example, miRNA
levels could be determined at 7 and 14 days following injury, or at
5, 10 and 15 days following injury. A return of miRNA levels to
normal may be indicative of recovery of the subject from the
TBI.
[0056] In some embodiments, a subject is determined to have
recovered from mTBI if the level of miR-425-5p and/or miR-502 is no
longer below a predetermined threshold or no longer decreased
relative to a reference.
[0057] In some embodiments, a subject is determined to have
recovered from moderate-to-severe TBI if the level of miR-21 and/or
miR-335 is no longer above a predetermined threshold or no longer
increased relative to a reference.
[0058] The diagnosis of a subject as suffering from a TBI, and in
particular diagnosis of mild TBI or moderate-to-severe TBI, may
facilitate in the determination of an appropriate treatment. The
present invention thus provides a test that enables healthcare
workers, such as physicians, clinicians, paramedics, and even
non-medical personnel (e.g. teachers, sports coaches, military
personnel) to decide on appropriate action for a subject suspected
of having a TBI. A subject determined as having a TBI may therefore
receive the most appropriate treatment as a result of a diagnosis
being made. The method of the invention may thus further comprise
directing appropriate therapy to a subject diagnosed with a
TBI.
[0059] A subject diagnosed with TBI may be further evaluated, e.g.
by CT scanning. In some embodiments, the subject is admitted to
hospital. In some embodiments, if moderate-to-severe TBI can be
ruled out, the subject may not need to be admitted to hospital for
evaluation. A subject diagnosed with moderate-to-severe TBI may be
admitted to hospital, or a specialist centre with neurotrauma
expertise.
[0060] A subject diagnosed with a TBI (particularly mTBI) outside a
hospital environment, for example, at a sporting event, during
combat or during play, may be removed from play or combat
immediately. The subject may subsequently be started on a graduated
return to play or combat.
[0061] In a further aspect, there is provided a method for
determining whether it is appropriate to administer to a subject a
therapy for alleviating TBI, the method comprising:
determining a level of at least one miRNA in a sample from the
subject; and determining whether or not it is appropriate to
administer a therapy for alleviating TBI, based on the level of the
at least one miRNA.
[0062] It will be appreciated that the step of administering the
therapy to the subject does not form a part of the claimed method,
unless specifically stated.
[0063] In some embodiments the method may further comprise
administering to the subject an appropriate treatment. In some
embodiments, the treatment may comprise a therapy for alleviating
TBI. Accordingly, the invention features methods of diagnosing and
treating TBI in a subject, the method comprising the steps of (a)
obtaining a sample (e.g., a sample of blood, plasma, urine, or
saliva) from the subject; (b) detecting one or more miRNAs
(selected from those described herein); diagnosing the patient as
having a TBI when the level(s) of the miRNA(s) differ from a
reference standard (as described herein); and administering a
treatment for the TBI.
[0064] In a further aspect, the invention provides a method of
determining an appropriate treatment to a subject suspected of
suffering from a TBI, the method comprising identifying whether or
not the subject has a TBI by determining a level of at least one
miRNA in a sample from the subject.
[0065] If a subject is identified as having a TBI, an appropriate
treatment may include one or more of the following: further
evaluating the subject, for example by further tests (e.g. verbal,
cognitive, motor and/or optical tests), CT and/or MRI scanning;
removing the subject from activity (e.g. the activity during which
the TBI was incurred); admitting the subject to hospital or a
specialist clinic; surgery; and administering a therapy for
alleviating TBI to the subject.
[0066] The therapy for alleviating TBI may include neuroprotective
drugs, e.g. drugs to treat cerebral swelling such as mannitol and
hypertonic saline, and/or other neuroprotective measures, such as
avoidance of hypertensive resuscitation and the use of
sedation.
[0067] In some embodiments, the subject may be subsequently
monitored to track their recovery, for example in a hospital or
clinic setting.
[0068] According to a further aspect of the invention, there is
provided a method of detecting and/or determining a level of a
target miRNA in a subject, the method comprising the steps of (a)
obtaining a sample from the subject; and (b) detecting and/or
determining the level of the target miRNA in the sample by
contacting the sample with a probe that is specific for the target
miRNA.
[0069] The sample may be any appropriate fluid or tissue sample
obtained from the subject, as defined above. In some embodiments,
the sample is blood, serum, plasma, urine or saliva.
[0070] In some embodiments, the method may comprise determining the
level of two or more target miRNAs in the sample.
[0071] According to a further aspect of the invention, there is
provided a therapy for alleviating TBI for use in a method of
treating a subject in need thereof, wherein said subject is
identified as having a TBI by determining a level of at least one
miRNA in a sample from the subject.
[0072] The step of determining the level of the target miRNA may
comprise contacting the sample with a substrate functionalized with
the probe, for example a chip comprising the probe. The substrate
or chip may conveniently include multiple probes, each specific for
a different target miRNA.
[0073] The subject may have suffered an injury, in particular a
head injury. The subject may be suspected as having a TBI. In some
embodiments, the sample is obtained no more than 72 hours, no more
than 48 hours, no more than 36 hours, no more than 24 hours, no
more than 12 hours, no more than 6 hours, no more than 4 hours, no
more than 2 hours or no more than 1 hour after injury.
[0074] In some embodiments, the method further comprises treating
the subject. The treatment may include one or more of the
following: further evaluating the subject, for example by further
tests (e.g. verbal, cognitive, motor and/or optical tests), CT
and/or MRI scanning; removing the subject from activity (e.g. the
activity during which the TBI was incurred); admitting the subject
to hospital or a specialist clinic; and administering a therapy for
alleviating TBI to the subject. In some embodiments, the treatment
comprises administering an effective amount of a neuroprotective
drug.
[0075] Thus, in yet a further aspect the invention provides a
method of treating TBI, the method comprising:
determining a level of at least one miRNA in a sample from the
subject; and if the level of the at least one miRNA is indicative
of mTBI, administering a treatment appropriate for mTBI; or if the
level of the at least one miRNA is indicative of m-sTBI,
administering a treatment appropriate for m-sTBI.
[0076] It will be appreciated by those skilled in the art that
different treatment pathways may be used for mTBI and m-sTBI.
[0077] An appropriate treatment for mTBI may include: removing the
subject from activity; treatment in situ or in the community;
further evaluating the subject in hospital without overnight
admission (typically mTBI patients are discharged with promptly
with head injury advice); or admission to hospital for a period of
observation (typically 1-2 days). The subject may be further
evaluated using tests (e.g. verbal, cognitive, motor and/or optical
tests). CT scanning is generally only required if certain
indications are present, including suspected skull fracture,
post-traumatic seizure, focal neurological deficit, repeated
vomiting, a GCS score of less than 13 on initial assessment (less
than 14 for children, or less than 15 for infants under 1 year), in
accordance with NICE guidelines.
[0078] An appropriate treatment for m-sTBI may include: MRI or CT
scanning (particularly within 1 hour of injury); admission to
hospital (which may include admission to intensive care and/or
transfer to a specialist clinic or major trauma centre with
neurosurgical facilities); neuromonitoring; surgery; administering
a therapy for alleviating TBI, such as administering
neuroprotective drugs, e.g. drugs to treat cerebral swelling such
as mannitol and hypertonic saline, and/or other neuroprotective
measures, such as avoidance of hypertensive resuscitation and the
use of sedation.
[0079] Thus, the present invention enables subjects with a TBI to
be quickly stratified into mTBI or m-sTBI, so that they may receive
the most appropriate treatment.
[0080] According to a further aspect of the invention, there is
provided a detection system for diagnosing and/or monitoring TBI,
the detection system comprising a sensor element comprising a
substrate functionalized with a probe specific for a target miRNA
The detection system can further comprise a detection device that
is capable of detecting the binding of a target miRNA to the
probe.
[0081] According to a yet further aspect of the invention, there is
provided a sensor element for use in a detection system for
diagnosing and/or monitoring TBI, the sensor element comprising a
substrate functionalized with a probe specific for a target
miRNA.
[0082] The sensor element may further comprise a sample addition
zone for receiving a sample (e.g. a fluid sample) thereon.
[0083] The probe is capable of selectively binding the miRNA of
interest. The substrate may be functionalized with a plurality of
probes. The probes may all be the same, or two or more different
probes may be provided. For example, in some embodiments, the
substrate may be functionalized with a first probe specific for a
first miRNA, and a second probe specific for a second miRNA. The
first and second probes may be grouped together, for example on
different portions of the sensor element.
[0084] In a further aspect of the invention, there is provided a
composition for use in a method of diagnosing and/or monitoring
traumatic brain injury (TBI) in a subject, the composition
comprising a probe specific for a target miRNA. The composition may
comprise any one of the listed miRNAs or with any plurality of the
listed miRNAs (e.g., two, three, four, or more of the listed
miRNAs).
[0085] In some embodiments, the target miRNA is selected from the
group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184,
miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335,
miR-126*, miR-193a-5p, miR-144*, miR-190, miR-194, miR-365,
miR-590-3p, miR-624, miR-625*, miR-671-3p, hsa-let-7c-5p,
hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p,
miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p,
miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-30a-5p; miR-107;
miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, miR-424-5p,
miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192,
miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301,
miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a,
miR-520D-3p and miR-629.
[0086] In some embodiments, the target miRNA is selected from the
group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184,
miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335,
hsa-miR-126*, miR-193a-5p, miR-144*, miR-190, miR-194, miR-365,
miR-590-3p, miR-624, miR-625*, and miR-671-3p. These microRNAs have
been found to be biomarkers expressed in all TBI patients (mild or
severe).
[0087] In some embodiments, the target miRNA is selected from the
group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184,
miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g and miR-335.
[0088] In some embodiments, the target miRNA is selected from the
group consisting of let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p,
miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p,
miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, and
miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p;
miR-324-5p; miR-652-3p.
[0089] In some embodiments, the target miRNA is selected from the
group consisting of miR-10a, miR-132, miR-223, miR-143, miR-148b,
miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152,
miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a,
miR-579, miR-642, miR-99a, miR-520D-3p and miR-629.
[0090] In some embodiments, the target miRNA is selected from the
group consisting of miR-425-5p, miR-502, miR-21 and miR-335.
[0091] The probe may comprise a biological molecule such as a
protein (e.g. an antibody) or a nucleic acid. In some embodiments,
the probe comprises a nucleic acid. The nucleic acid may comprise a
sequence which is at least 70%, at least 75%, at least 80%, at
least 85%, at least 90% or at least 95% identical to a sequence
which is the complement of the full-length sequence of the target
miRNA. In some embodiments, the nucleic acid comprises a sequence
which is 100% identical to a sequence which is the complement of
the sequence of the target miRNA (i.e. the receptor comprises a
nucleic acid sequence which is the exact complement of the target
miRNA sequence).
[0092] The probes may be attached to a surface of the substrate by
any suitable means, such as by coupling chemistry known to those
skilled in the art. In some embodiments, each probe is attached to
a surface of the substrate via a linker. In some embodiments, the
probe comprises a moiety for immobilizing the probe on the
substrate, or for attaching the probe to a linker immobilized on
the substrate.
[0093] Alternatively or in addition, the probe may comprise a
detectable label. The detectable label may be, for example,
radioactive, fluorescent, luminescent, or antibody-based (e.g., it
may constitute a conventional tetrameric antibody or a detectable
fragment thereof).
[0094] The substrate of the sensor element may be formed from any
suitable material. In some embodiments, the substrate comprises or
is formed from metal, plastic, glass, silica, silicon, graphite,
graphene, or any combination thereof. In some embodiments, the
substrate comprises multiple layers. For example, a substrate may
be prepared by forming a surface or layer of graphene on a layer of
silicon carbide or silica. The graphene surface may be chemically
modified, for example to graphene-oxide (GO) or graphene-amine
(GA). Methods for forming graphene layers, such as epitaxial growth
and sublimation growth, will be known to those skilled in the
art.
[0095] Conveniently, probes comprising or constituted by a nucleic
acid can be attached to a GO surface via a linker, using an amide
coupling reagent (e.g.
(O-(7-azabenzotriazole-1-yl)-N,N,N,N'-tetramethyluronium
hexafluorophosphate (HATU)). A sensor element comprising a surface
functionalized with a nucleic acid probe can then be used to
selectively detect its complementary miRNA.
[0096] Suitable linkers may comprise an aniline moiety (or a
derivative thereof), a benzoic acid moiety (or a derivative
thereof) or an ethendiamine moiety (or a derivative thereof). An
aniline linker may be formed by attaching a nitrobenzene molecule
(or derivative) to a graphene surface (e.g. using a diazonium
salt), and reducing the nitrobenzene to aniline. The amine group of
the aniline may then be used to attach to the probe. Similarly, a
diazonium salt (e.g. 4-benzoic acid diazonium tetrafluoroborate)
can be used to attach a benzoic acid or benzoic acid derivative to
a graphene surface. An ethanediamine moiety may be attached to
carboxylated graphene or graphene oxide.
[0097] The sensor element may be comprised within a test strip. The
test strip may be disposable.
[0098] The detection device may be configured to detect the binding
of a target miRNA to the receptor by any suitable means known to
those skilled in the art, for example by detecting changes in
electrical impedance, hydrogen ion concentration, or conformational
changes resulting from hybridisation.
[0099] The detection device may further include a user interface to
output data to a user.
[0100] In some embodiments, the detection device includes a
database of treatment information. The device may be capable of
identifying suitable treatment options from the database depending
on the levels of the or each miRNA of interest. The treatment
information may be provided to the user via the user interface.
[0101] Conveniently, the detection device may be portable, e.g.
hand-held. The detection device may comprise a data storage unit
for storing miRNA levels and other information relating to the
subject. In some embodiments, the device comprises a data
communication means for communicating data to other devices. For
example, the device may communicate data wirelessly through WiFi,
3G, 4G, Bluetooth, or through a mobile app. This may conveniently
enable the data to be easily accessed by medical professionals if
necessary.
[0102] It is thus envisaged that the detection device of the
invention provides an affordable, portable, point of care means for
diagnosing and monitoring TBI non-invasively. The device may be
used by ambulance crews, the military, schools, sports clubs and
healthcare professionals, enabling the correct assessment and
triage of patients suspected to have a TBI.
[0103] In a further aspect there is provided a kit for use in the
present methods. The kit may comprise at least probe (e.g. a
protein, such as an antibody, or a nucleic acid) which is capable
of selectively binding the miRNA of interest. In some embodiments,
the kit comprises an array comprising a plurality of probes. In
some embodiments, the at least one probe is a primer for carrying
out PCR. The kit may further comprise instructions for use, for
example instructions for use in the diagnosis and/or monitoring of
TBI. The kit may further comprise suitable buffers and reagents,
such as amplification primers and enzymes (e.g. DNA polymerase,
reverse transcriptase for conversion of miRNA to cDNA).
[0104] It will be appreciated that statements made herein in
relation to any aspect of the vention may equally apply to any
other aspect of the invention, as appropriate.
DETAILED DESCRIPTION OF THE INVENTION
[0105] Embodiments of the invention will now be described by way of
example and with reference to the accompanying Figures:
[0106] FIG. 1: miR-425-5p and miR-502 expression in 3 different
categories of trauma and HV MiR-425-5p and miR-502 expression in 10
HV, 10 mTBI+EC (1 day), 10 mTBI+EC (15 days), 10 EC (1 day), 10 EC
(15 days), 10 sTBI+EC (1 day) and 10 sTBI+EC (15 days) patients,
detected by qRT-PCR analysis. MiR-425-5p expression was found to be
remarkably decreased in mTBI+EC (1 day) compared to HV (p<0.01),
mTBI+EC (15 dys) (p<0.001) and sTBI+EC (1 day) (p<0.01) (FIG.
1A). MiR-502 expression was found to be remarkably decreased in
mTBI+EC (1 day) compared to HV (p<0.05), mTBI+EC (15 dys)
(p<0.01) and sTBI+EC (1 day) (p<0.05) (FIG. 1B).
[0107] FIG. 2: miR-21 and miR-335 expression in 3 different
categories of trauma and HV MiR-21 and miR-335 expression in 10 HV,
10 mTBI+EC (1 day), 10 mTBI+EC (15 days), 10 EC (1 day), 10 EC (15
days), 10 sTBI+EC (1 day) and 10 sTBI+EC (15 days) patients,
detected by qRT-PCR analysis. MiR-21 expression was found to be
significantly up-regulated in sTBI+EC (1 day and 15 days) compared
to HV (p<0.01) (FIG. 2A). MiR-335 expression was found to be
remarkably up-regulated in sTBI+EC (1 day) compared to HV
(p<0.001), EC (15 days) (p<0.001) and mTBI+EC (1 day)
(p<0.05) (FIG. 2B).
[0108] FIG. 3: Time course of miR-425-5p (FIG. 3A) and miR-502
(FIG. 3B) expression in 3 different categories of trauma and HV
[0109] MiR-425-5p and miR-502 expression in 30 HV, 30 mTBI+EC, 30
EC, 30 sTBI+EC patients at different time points from injury (T0,
T4-12h, T48-72h, 15 days) detected by qRT-PCR analysis. MiR-425-5p
expression was found to be remarkably decreased in mTBI+EC at T0
and T4-12h compared to HV, sTBI+EC and EC (p<0.05). MiR-502
expression was found to be remarkably decreased in mTBI+EC at T0
and T4-12h compared to HV, sTBI+EC and EC (p<0.05). P values
were determined by Tukey's post-hoc test.
* significantly different from HV
[0110] FIG. 4: Time course of miR-21 (FIG. 4A) and miR-335 (FIG.
4B) expression in 3 different categories of trauma and HV
[0111] MiR-21 and miR-335 expression in 30 HV, 30 mTBI+EC, 30 EC,
and 30 sTBI+EC patients at different time points from injury (T0,
T4-12h, T48-72h, 15 days) detected by qRT-PCR analysis. MiR-21
expression was found to be significantly up-regulated in sTBI+EC at
T4-12h, T48-72h and 15 days compared to HV (p<0.01). MiR-335
expression was found to be remarkably up-regulated in sTBI+EC at
T0, T4-12h, T48-72h and 15 days compared to HV and mTBI+EC
(p<0.001) but not EC only. P values were determined by Tukey's
post-hoc test.
* significantly different from HV
EXAMPLES
[0112] As miRNAs are emerging as promising biomarkers in a range of
different pathologies, the present inventors sought to explore
their role in TBI.
Example 1
[0113] Materials and Methods
[0114] Patients and Samples Collection
[0115] Study participants were recruited from the Surgical
Reconstruction and Microbiology Research Centre (SRMRC) at Queen
Elizabeth Hospital of Birmingham (UK) as part of Brain Biomarkers
after Trauma (The Golden Hour Study) study (Ethics Ref.
13/WA/0399).
[0116] First, we performed screening of 754 miRNAs in 5 mTBI with
extra-cranial injury (EC) patients, 5 sTBI+EC injury patients and
healthy volunteers (HV) at 1 day and 15 days from injury with the
aim to select specific candidate biomarkers able to discriminate
mild from severe TBI and predict the recovery of mTBI after 15
days. Based on this information (Table 2), it was then possible to
confirm the results study in an enlarged cohort of patients of 40
individuals grouped in 4 different categories: HV (n=10), EC
(n=10), mTBI+EC (n=10), sTBI+EC (n=10). Healthy volunteers were
consented and enrolled in the RECOS study. EC injury patients had
radiographically-confirmed fractures, no head trauma, no infection,
no history of neurological or psychiatric disorders and no alcohol
or drug dependency. Mild TBI with EC included those with a
non-penetrating head trauma and Glasgow Coma Scale (GCS) score
>13. Severe TBI with EC included patients with GCS score of 8 or
below. All patients were gender and age matched to HVs.
[0117] Sample Processing
[0118] Peripheral blood samples were taken at 1 day and 15 days
from injury in each patient. The blood samples were processed for
serum isolation within 2h after withdrawal. Whole blood was left to
stand for about 30' at room temperature before being centrifuged at
3000 rpm for 10' at 4.degree. C. Serum was divided into aliquots
and stored at -80.degree. C. until analysis.
[0119] RNA Isolation, Reverse Transcription and miRNAs Profiling by
TaqMan Low Density Array (TLDA)
[0120] Initial screening (discovery set) were performed on 5
mTBI+EC and 5 sTBI+EC patients, which were compared to HV at the
two different time points (1 day and 15 days from the injury). The
serum of these patients was used to profile the transcriptome of
754 miRNAs. Serum samples were centrifuged at 2000 rpm for 10' to
pellet and remove any circulating cell or debris. MiRNAs were
extracted from 400 .mu.l of serum samples by using Qiagen miRNeasy
mini kit (Qiagen, GmbH, Hilden, Germany), according to Qiagen
supplementary protocol for purification of small RNAs from serum
and plasma and finally eluted in 30 .mu.l volume of RNase free
water. The concentration and purity of the resulting RNA was
determined with a ND-1000 UV-Vis Spectrophotometer (NanoDrop). 20
ng of serum RNAs was retrotranscribed and pre-amplified, according
to the manufacturer's instructions. Pre-amplified products were
loaded onto TLDAs, TaqMan Human MicroRNA Array v3.0 A and B
(Applied Biosystems LifeTechnologies.TM.). PCRs on TLDAs were
performed on 7900HT Fast RealTime PCR System (Applied Biosystem,
LifeTechnologies.TM.).
[0121] Data Analysis
[0122] To obtain an accurate miRNA profiling, we used the global
median normalization method. Similar to microarray analysis, Ct
values from each sample were normalized to the median Ct of the
array. Moreover, by computing the Pearson correlation among the Ct
medians and means of each array and Ct of each miRNA, we identified
two miRNAs that showed an expression profile close to the median
and mean of TLDAs, i.e., miR-331 and miR-223*. These miRNAs were
also confirmed to be among the most stable in TLDAs by applying two
different methods: DataAssistv.3software (AppliedBiosystem Life
Technologies.TM.) and geNorm Algorithm. Accordingly, miR-331 and
miR-223* were used as reference genes for validation by single
TaqMan assays. Expression fold changes were calculated by the
2-.DELTA..DELTA.CT method. Differentially expressed miRNAs (DE
miRNAs) were identified by Significance of Microarrays Analysis
(SAM) computed by Multi experiment viewer v4.8.1, applying a
two-class unpaired test among .DELTA.Cts and using a p-value based
on 100 permutations; imputation engine: K-nearest neighbours (10
neighbours); false discovery rate<0.15 was used as correction
for multiple comparisons. We accepted as reliable only DE miRNAs
concordant by using all endogenous controls.
[0123] Single TaqMan Assays
[0124] Ten differentially expressed miRNAs were chosen from the
arrays as potential candidate biomarkers with the aim to
discriminate mild from severe TBI and to monitor the recovery of
mild. These candidates were used to validate the data in an
enlarged cohort of 30 patients (validation set) grouped in 3
different categories (mTBI+EC, sTBI+EC and EC only) and 10 controls
(HV) at two different time points (1 and 15 days from injury) by
single TaqMan assays (Applied Biosystems, Life Technologies.TM.).
Samples were extracted and retrotrascribed as described above and
RT-qPCR analysis was performed in Bio-Rad iQ5 Real-time PCR
Detection System (Bio-Rad, CA, USA). Expression fold changes were
calculated by the 2-.DELTA..DELTA.CT method.
[0125] Statistical Analysis
[0126] The data were check for normal distribution and transformed
to perform parametric tests. Comparisons across groups at each time
and within groups over time were performed by the one-way analysis
of variance and Tukey's post-hoc test on transformed data. A
receiver operating characteristic analysis was utilised to
calculate sensitivity and specificity of each biomarker in
diagnosing either mTBI or sTBI expressed as area under the curve
(AUC). All analyses were carried on SPSS v.20 (IBM). Differences
were considered as statistically significant at p-value
<0.05.
[0127] Results
[0128] Expression Profiles by TaqMan Low Density Arrays (TLDA)
[0129] From 754 screenable miRNAs of TLDA, we identified ten
circulating miRNAs at 1 day and 13 at 15 days in mTBI+EC, 19 at 1
day and 22 at 15 days in sTBI+EC differentially expressed (Table
2). From this list, hsa-miR-126*, miR-193a-5p, miR-144*, miR-190,
miR-194, miR-365, miR-590-3p, miR-624, miR-625* and miR-671-3p were
excluded for further analysis because they were expressed in most
of the patients, hence not suitable candidate biomarkers for mild
or severe trauma only. However, the above microRNAs can identify
TBI of any severity and are therefore useful TBI biomarkers. On the
other hand, miR-184, miR-301b, miR-502 and miR-505 uniquely and
differentially expressed in mTBI+EC at 1 day, were selected as
early candidate biomarkers of mTBI. In addition, miR-203,
miR-425-5p, miR-654-3p and miR-655 differentially expressed at 15
days post mTBI+EC were selected as candidate biomarkers able to
track the recovery of mTBI.
[0130] Finally, two miRNAs, miR-21 and miR-335, constantly
expressed at both time points in sTBI+EC, were selected for further
studies.
TABLE-US-00002 TABLE 2 Fold change of microRNAs differentially
expressed in 5 mTBI + EC (1 and 15 days) and 5 sTBI + EC (1 and 15
days) patients, compared to 5 HV and detected by TLDA. mTBI + EC 1
day mTBI + EC 15 days sTBI + EC sTBI + EC 15 days VS HV VS HV 1 day
VS HV VS HV hsa-miR- 0.1383 hsa-miR- 24.901 hsa-let-7g 0.3250
hsa-miR- 14.979 184 193a-5p 130a hsa-miR- 0.1278 hsa-miR- 20.946
hsa-miR- 11.953 hsa-miR- 9.8921 190 194 10a 152 hsa-miR- 0.0798
hsa-miR- 4.1763 hsa-miR- 8.1875 hsa-miR- 0.0277 425-5p 203 132 190
hsa-miR- 0.0538 hsa-miR- 3.7087 hsa-miR- 26.193 hsa-miR- 13.655 502
365 143 193a-5p hsa-miR- 7.7696 hsa-miR- 3.0166 hsa-miR- 20.095
hsa-miR- 12.301 505 425-5p 148b 194 hsa-miR- 0.1570 hsa-miR- 0.0878
hsa-miR- 26.806 hsa-miR-21 12.662 126* 654-3p 18a hsa-miR- 0.2758
hsa-miR- 0.0756 hsa-miR- 0.0634 hsa-miR- 18.977 144* 655 190 27b
hsa-miR- 0.3842 hsa-miR- 4.0584 hsa-miR- 0.2245 hsa-miR- 21.954
590-3p 671-3p 192 301 hsa-miR- 0.0836 hsa-miR- 0.2978 hsa-miR-
29.785 hsa-miR- 93.099 624 126* 193a-5p 326 hsa-miR- 0.0435
hsa-miR- 0.2645 hsa-miR-21 7.1654 hsa-miR- 45.050 301b 144* 335
hsa-miR- 0.3035 hsa-miR- 4.6799 hsa-miR- 37.699 590-3p 223 345
hsa-miR- 0.1643 hsa-miR- 37.192 hsa-miR- 37.295 624 335 361
hsa-miR- 0.1521 hsa-miR- 5.5771 hsa-miR- 77.373 625* 365 422a
hsa-miR- 0.1294 hsa-miR- 4.7203 429 579 hsa-miR- 29.527 hsa-miR-
41.515 618 642 hsa-miR-95 20.788 hsa-miR- 8.4425 671-3p hsa-miR-
0.2759 hsa-miR- 12.218 144* 99a hsa-miR- 0.0304 hsa-miR- 0.2905 624
144* hsa-miR- 0.0981 hsa-miR- 0.2579 625* 520D-3p hsa-miR- 0.4188
590-3p hsa-miR- 0.1575 625* hsa-miR- 0.2793 629
[0131] Single TaqMan Assay for Candidate Biomarkers of mTBI
[0132] In order to validate these findings, we subsequently tested
the expression of selected miRNAs in three separate and independent
groups (10 mTBI+EC, 10 sTBI+EC, 10 EC) at the two chosen time
points (1 and 15 days from injury) by using single TaqMan assays.
The results were compared to 10 HV. The fold changes were
calculated by the 2-.DELTA..DELTA.CT method, using miR-331 and
miR-223* as reference genes.
[0133] Among the candidate biomarkers of mTBI at both time points
(miR-184, miR-301b, miR-502, miR-505, miR-203, miR-425-5p,
miR-654-3p and miR-655), two showed interesting results and were
significantly and differentially expressed in the three different
categories compared to HV. In particular, miR-425-5p and miR-502
showed a similar trend (FIG. 1). They were both significantly
downregulated in mTBI+EC (mean of 0.387.+-.0.201 and
0.314.+-.0.146) respectively at 1 day from injury compared to HV
(p<0.001), EC (p<0.001) and sTBI+EC (p<0.001). At 15 days
from mild injury, miR425-5p and miR-502 returned back to normal
levels (0.886.+-.0.310 and 1.157.+-.0.258). The expression of
miR-425-5p and miR-502 was also found similar to HV in EC samples
at both 1 day and 15 days from injury, thus suggesting that these
two biomarkers are differentially expressed in brain injury
patients only. Moreover, neither of them showed any significant
difference in sTBI+EC compared to HV at both time points.
Therefore, miR-425-5p and miR-502 could be considered the most
promising candidate biomarkers for the early diagnosis and
monitoring of mTBI at 15 days after trauma. AUCs for these
biomarkers are shown in Table 3.
[0134] Single TaqMan Assay for Candidate Biomarkers of sTBI
[0135] miR-21 and miR-335 were analysed as potential biomarkers of
sTBI since they both appeared upregulated at both time points of
sTBI+EC in the initial screening. They showed to be strong
candidates in the second dataset of patients as well (FIG. 2).
Mir-21 was significantly upregulated at both time points in sTBI
with EC (7.106.+-.4.192 and 4.012.+-.1.577) with respect to HV (p,
0.001), EC (p<0.001) and mTBI (p<0.001). No significant
differences were found in the remaining categories compared to HV.
miR-335 showed upregulation in sTBI+EC and at both time points
(16.824.+-.14.195 and 12.324.+-.8.931, respectively). On day 1,
there this group were significantly different from controls
(p=0.001) and mTBI+EC (p=0.031) but not EC. Interestingly, a
significant upregulation in EC patients was found at 1 day
(7.951.+-.4.870), but not at 15 days from injury (1.260.+-.0.531).
For this reason, on day 15, miR335 was significantly higher in the
sTBI+EC group with respect to HV (p=0.002), EC (p=0.007) and
mTBI+EC (p=0.001). miR-335 did not show any significant difference
in mTBI+EC at both time points compared to HV. AUCs for these
biomarkers are also shown in Table 3.
TABLE-US-00003 TABLE 3 Area under the curve Asymptotic 95%
Confidence Interval Variable(s) Area Lower Bound Upper Bound
miR-502 1 day .993 .972 1.000 miR-502 15 days Not Significant -- --
miR-425-5p 1 day .994 .977 1.000 miR-425-5p 15 days Not Significant
-- -- miR-21 1 day .979 .934 1.000 miR-21 15 days .975 .929 1.000
miR-335 1day .758 .592 .921 miR-335 15 days .957 .884 1.000
[0136] Discussion
[0137] The present study investigated if changes in the levels of
miRNAs can be applied to the diagnosis of TBI, and evaluating its
severity. Four miRNAs were identified as being differentially
expressed in TBI; miR-425-5p, miR 502, miR-21 and miR-335.
[0138] miR-425-5p showed significant results at day 1 in mTBI+EC
compared to the HV and similar results were obtained in all other
categories. Its downregulation within the first 24h from the mild
injury and the its return back to normal levels after 15 days,
makes miR-425-5p a suitable candidate biomarker of mild trauma.
[0139] miR-502 was also found to be differentially expressed in
mTBI+EC. The trend of this miRNA was very similar to miR-425-5p. It
showed specificity for brain injured patients and could also be
used to track recovery, since it returns back to normal value after
15 days form the mild injury.
[0140] Following sTBI, 2 miRNAs (miR-21, miR-335) were noted to be
expressed at both 1 and 15 days and were thus selected as potential
biomarkers for sTBI. miR-21 and miR-335 were significantly
up-regulated at both time points when compared with controls in
sTBI+EC. Therefore, the overexpression confirmed the results of the
array and showed the potential of these molecules as biomarkers of
sTBI.
[0141] The selected panel of miRNAs have the potential to diagnose
TBI accurately and enable the stratification of patients according
to severity which, in turn, allows the delivery of the most
appropriate treatment.
Example 2
[0142] Patients and Sample Collection
[0143] Study participants were recruited from the Surgical
Reconstruction and Microbiology Research Centre (SRMRC) at Queen
Elizabeth Hospital of Birmingham (UK) as part of SIR (The Steroids
and Immunity from injury through to Rehabilitation) study (Ethics
Ref. 11/SW/0177), ReCoS (The REpetitive COncussion in Sport) study
(Ethics Ref. 11-0429AP28) and Golden Hour study (Ethics Ref.
13/WA/0399). Written informed consent was received from
participants or valid proxy (family or a professional not directly
involved in the study) prior to inclusion in the study.
[0144] The second dataset of samples used serum samples from a
total of 120 individuals grouped in 4 different categories: HV
(n=30), EC (n=30), mTBI+EC (n=30), sTBI+EC (n=30) and blood was
taken at different time points (T0-1h, T4-12h, T48-72h, 15 days) in
each patient. Healthy volunteers were consented and enrolled in the
ReCoS study. EC injury patients had radiographically-confirmed
orthopedic fractures, no head trauma, no infection, no history of
neurological or psychiatric disorders and no alcohol or drug
dependency. Mild TBI with EC included those with a non-penetrating
head trauma and Glasgow Coma Scale (GCS) score .gtoreq.13. Severe
TBI with EC included patients with GCS.ltoreq.8.
[0145] Sample Processing
[0146] The blood samples were processed for serum isolation within
2h after withdrawal. Whole blood was left to stand for about 30' at
room temperature before being centrifuged at 3000 rpm for 10' at
4.degree. C. Serum was divided into aliquots and stored at
-80.degree. C. until analysis.
[0147] RNA isolation, data analysis, assays and statistical
analysis were carried out as described in Example 1.
[0148] Results
[0149] Single TaqMan Assay for Candidate Biomarkers of mTBI
[0150] In order to validate the findings of Example 1, the
expression of selected miRNAs in 3 separate and independent groups
(30 mTBI+EC, 30 sTBI+EC, 30 EC) was measured at different time
points (T0, T4-12h, T48-72h and 15 days from the injury) by using
single TaqMan assays. The results were compared to 10 HV. The fold
changes were calculated by the 2-.DELTA..DELTA.CT method, using
miR-331 and miR-223* as reference.
[0151] Among the candidate biomarkers of mTBI at both time points
(miR-184, miR-502, miR-505, miR-301b, miR-203, miR-425-5p,
miR-654-3p and miR-655), only two showed interesting results and
were significantly and differentially expressed in the 3 different
categories compared to HV. Specifically, miR-425-5p and miR-502
showed a similar trend (FIG. 3). They were both significantly
downregulated in mTBI+EC, miR-425-5p at T0-1h (p=0.01845), and at
T4-12h (p=0.01962) respectively compared to HV, or EC and sTBI+EC
(p<0.05), and miR-502 at T0-1h and at T4-12h compared to HV
(p=0.02538 and p=0.03718 respectively), or EC and sTBI+EC
(p<0.01). After 48h from mild injury, miR425-5p and miR-502
returned back to normal levels. The expression of miR-425-5p and
miR-502 was also found in EC group at a comparable level to HV,
thus suggesting that these two biomarkers are downregulated in
brain injury patients only. Moreover, neither of them showed any
significant downregulation in sTBI+EC compared to HV at all time
points. Therefore, miR-425-5p and miR-502 could be considered the
most promising candidate biomarkers for the early diagnosis and
monitoring of mTBI. AUCs for these biomarkers at the most relevant
time points, are shown in Table 4.
[0152] Single TaqMan Assay for Candidate Biomarkers of sTBI
[0153] MiR-21 and miR-335 were analysed as potential biomarkers of
sTBI since they both appeared upregulated at both time points of
sTBI+EC in the initial screening. They showed to be strong
candidates in the second dataset of patients also (FIG. 4). Mir-21
was significantly upregulated in sTBI with EC at all time points
after 4h from injury with respect to HV, EC and mTBI+EC (p=0.00306
at T4-12h, p=0.00844 at T48-72h and p=0.00077 at 15 days). No
significant differences were found in the remaining categories
compared to HV. MiR-335 showed upregulation in sTBI+EC and at all
time points compared to HV (p=0.00109 at T0-1h, p=0.00284 at
T4-12h, p=0.00012 T48-72h and p=0.01284 at 15 days) and mTBI+EC but
not significant upregulation was found compared to EC. AUCs for
these biomarkers are also shown in Table 4.
TABLE-US-00004 TABLE 4 Area under the curve of the four candidate
biomarkers of TBI at different time points. Only most relevant time
points are shown. Asymptotic 95% Confidence Interval Lower Upper
Variable(s) Area Sig Bound Bound miR-425-5p T0 mTBI vs HV 1 .002 1
1 mTBI vs sTBI .911 .020 .778 1.000 mTBI vs EC .950 .01 .860 1.000
miR-502 T0 mTBI vs HV 1 .001 1.000 1.000 mTBI vs sTBI .990 .001
.960 1.000 mTBI vs EC .990 .001 .940 1.000 miR-21 T4-12h sTBI vs HV
.961 .003 .880 1.000 sTBI vs mTBI .960 .001 .870 1.000 sTBI vs EC
.900 .004 .740 1.000 miR-335 T0 sTBI vs HV .990 .000 .960 1.000
sTBI vs mTBI .780 .023 .590 .960 sTBI vs EC .500 1 .240 0.780
[0154] Discussion
[0155] This study validated the previous finding that changes in
the levels of miRNAs can be applied to the diagnosis of TBI, and
evaluating its severity. The study confirmed that the following
four miRNAs are differentially expressed in TBI; miR-425-5p, miR
502, miR-21 and miR-335.
[0156] miR-425-5p showed significant results at T0 and T4-12h in
mTBI+EC compared to the HV and similar results were obtained in all
other categories. Its downregulation return back to normal levels
after T48-72h, confirms that miR-425-5p a suitable candidate
biomarker of mild trauma.
[0157] miR-502 was also confirmed to be differentially expressed in
mTBI+EC. The trend of this miRNA was very similar to miR-425-5p. It
showed specificity for brain injured patients and could also be
used to track recovery, since it returns back to normal value after
T48-72h form the mild injury.
[0158] Following sTBI, 2 miRNAs (miR-21, miR-335) were noted to be
expressed at all time points analysed and were thus confirmed as
potential biomarkers for sTBI. miR-21 and miR-335 were
significantly up-regulated when compared with controls in sTBI+EC.
Therefore, the overexpression confirmed the potential of these
molecules as biomarkers of sTBI.
Example 3
[0159] Saliva samples were collected from concussed professional
athletes after 2-3 days from injury and the microRNAs present in
the saliva was analysed. The athletes were diagnosed clinically as
having mTBI.
[0160] Materials and Methods
[0161] MicroRNAs were analysed using nCounter technology
(nanoString Technologies.RTM.), which uses molecular "barcodes" and
microscopic imaging to detect and count up to several hundred
unique transcripts in one hybridization reaction. Each colour-coded
barcode is attached to a single target-specific probe corresponding
to a microRNAs of interest.
[0162] Analysis was carried out according to the manufacturer's
protocol which includes the following steps:
[0163] Hybridization: The technology employs two .about.20-base
probes per microRNA that hybridize in solution. A reporter probe
carries the signal, while a capture probe allows the complex to be
immobilized for data collection.
[0164] Purification and Immobilization: After hybridization, the
excess probes are removed and the probe/target complexes are
aligned and immobilized in the nCounter Cartridge.
[0165] Data Collection: Sample cartridges are placed in a digital
analyzer instrument for data collection. Colour codes on the
surface of the cartridge are counted and tabulated for each target
molecule.
[0166] Results
[0167] Table 5 below is a list of microRNAs which were found to be
significantly and differentially expressed in concussed athletes
compared to healthy volunteers. The table shows the fold change in
expression of the microRNAs in the patients compared to the control
group. The fold changes were calculated using miR-23a-3p and
miR-148b-3p as reference genes.
TABLE-US-00005 TABLE 5 microRNA Fold change hsa-let-7c-5p 2.80
hsa-let-7i-5p 1.82 hsa-miR-142-3p 1.41 hsa-miR-148a-3p 1.72
hsa-miR-15b-5p 2.30 hsa-miR-16-5p 2.32 hsa-miR-181a-5p 1.54
hsa-miR-20a-5p + hsa-miR-20b-5p.sup..sctn. 2.16 hsa-miR-221-3p 2.90
hsa-miR-24-3p 1.94 hsa-miR-27b-3p 3.45 hsa-miR-29a-3p 4.03
hsa-miR-29c-3p 1.44 hsa-miR-424-5p 2.88 hsa-miR-30a-5p 2.16
hsa-miR-107 1.72 hsa-miR-135b-5p 1.97 hsa-miR-199b-5p 1.88
hsa-miR-324-5p 5.61 hsa-miR-652-3p 3.43 .sup..sctn.the values for
miR-20a-5p and miR-20b-5p were combined due to the nCounter
technology not being able to distinguish between them.
[0168] Discussion
[0169] This study shows that microRNAs present in saliva are an
indicator of concussion/mTBI. This is significant because saliva is
more easily obtained than blood, and thus detection of salivary
microRNAs offers a rapid and convenient means for diagnosing TBI,
particularly pitch-side.
Sequence CWU 1
1
69122RNAHomo sapiens 1uagcuuauca gacugauguu ga 22223RNAHomo sapiens
2aaugacacga ucacucccgu uga 23321RNAHomo sapiens 3auccuugcua
ucugggugcu a 21423RNAHomo sapiens 4ucaagagcaa uaacgaaaaa ugu
23523RNAHomo sapiens 5cagugcaaug auauugucaa agc 23622RNAHomo
sapiens 6uggacggaga acugauaagg gu 22722RNAHomo sapiens 7cgucaacacu
ugcugguuuc cu 22822RNAHomo sapiens 8gugaaauguu uaggaccacu ag
22922RNAHomo sapiens 9uaugucugcu gaccaucacc uu 221022RNAHomo
sapiens 10auaauacaug guuaaccucu uu 221121RNAHomo sapiens
11gccccugggc cuauccuaga a 211222RNAHomo sapiens 12cguguauuug
acaagcugag uu 221322RNAHomo sapiens 13ugagguagua guuuguacag uu
221421RNAHomo sapiens 14cauuauuacu uuugguacgc g 211522RNAHomo
sapiens 15ugggucuuug cgggcgagau ga 221622RNAHomo sapiens
16ggauaucauc auauacugua ag 221721RNAHomo sapiens 17cuauauauca
aacauauucc u 211822RNAHomo sapiens 18uguaacagca acuccaugug ga
221922RNAHomo sapiens 19uaaugccccu aaaaauccuu au 222021RNAHomo
sapiens 20uaauuuuaug uauaagcuag u 212122RNAHomo sapiens
21uaguaccagu accuuguguu ca 222222RNAHomo sapiens 22gacuauagaa
cuuucccccu ca 222321RNAHomo sapiens 23uccgguucuc agggcuccac c
212422RNAHomo sapiens 24ugagguagua gguuguaugg uu 222522RNAHomo
sapiens 25ugagguagua guuugugcug uu 222623RNAHomo sapiens
26uguaguguuu ccuacuuuau gga 232722RNAHomo sapiens 27ucagugcacu
acagaacuuu gu 222822RNAHomo sapiens 28uagcagcaca ucaugguuua ca
222922RNAHomo sapiens 29uagcagcacg uaaauauugg cg 223023RNAHomo
sapiens 30aacauucaac gcugucggug agu 233123RNAHomo sapiens
31uaaagugcuu auagugcagg uag 233223RNAHomo sapiens 32caaagugcuc
auagugcagg uag 233323RNAHomo sapiens 33agcuacauug ucugcugggu uuc
233422RNAHomo sapiens 34uggcucaguu cagcaggaac ag 223521RNAHomo
sapiens 35uucacagugg cuaaguucug c 213622RNAHomo sapiens
36uagcaccauc ugaaaucggu ua 223722RNAHomo sapiens 37uagcaccauu
ugaaaucggu ua 223822RNAHomo sapiens 38uguaaacauc cucgacugga ag
223923RNAHomo sapiens 39agcagcauug uacagggcua uca 234023RNAHomo
sapiens 40uauggcuuuu cauuccuaug uga 234123RNAHomo sapiens
41cccaguguuu agacuaucug uuc 234223RNAHomo sapiens 42cgcauccccu
agggcauugg ugu 234321RNAHomo sapiens 43aauggcgcca cuaggguugu g
214422RNAHomo sapiens 44cagcagcaau ucauguuuug aa 224523RNAHomo
sapiens 45uacccuguag auccgaauuu gug 234622RNAHomo sapiens
46uaacagucua cagccauggu cg 224722RNAHomo sapiens 47ugucaguuug
ucaaauaccc ca 224821RNAHomo sapiens 48ugagaugaag cacuguagcu c
214922RNAHomo sapiens 49ucagugcauc acagaacuuu gu 225023RNAHomo
sapiens 50uaaggugcau cuagugcaga uag 235121RNAHomo sapiens
51cugaccuaug aauugacagc c 215222RNAHomo sapiens 52uaauacuguc
ugguaaaacc gu 225323RNAHomo sapiens 53aaacucuacu uguccuucug agu
235421RNAHomo sapiens 54ucaauaaaug ucuguugaau u 215522RNAHomo
sapiens 55cagugcaaug uuaaaagggc au 225623RNAHomo sapiens
56agguucugug auacacuccg acu 235721RNAHomo sapiens 57uucacagugg
cuaaguucug c 215823RNAHomo sapiens 58cagugcaaua guauugucaa agc
235920RNAHomo sapiens 59ccucugggcc cuuccuccag 206022RNAHomo sapiens
60gcugacuccu aguccagggc uc 226122RNAHomo sapiens 61uuaucagaau
cuccaggggu ac 226222RNAHomo sapiens 62acuggacuua gggucagaag gc
226323RNAHomo sapiens 63uucauuuggu auaaaccgcg auu 236422RNAHomo
sapiens 64gucccucucc aaaugugucu ug 226522RNAHomo sapiens
65aacccguaga uccgaucuug ug 226622RNAHomo sapiens 66aaagugcuuc
ucuuuggugg gu 226721RNAHomo sapiens 67uggguuuacg uugggagaac u
216821RNAHomo sapiens 68aucacauugc cagggauuuc c 216922RNAHomo
sapiens 69ucagugcauc acagaacuuu gu 22
* * * * *