“MicroRNAs as

Biomarkers in
Amyotrophic Lateral
Sclerosis”
Paper published by the Department of Medical, Surgical, and
Neurological Sciences, University of Siena in Siena, Italy

Claudia Ricci, Carlotta Marzocchi and Stefania Battestini

Published 20 November 2018

Outline

Synopsis Slide 3

Background Slides 4-11

Methodology Slides 12-20

Data & Analysis Slides 21-25

Conclusion Slides 26-27

Further Studies Slides 28-29

Synopsis
● ALS is an incurable
neurodegenerative disease. Current
treatments only work to attempt to
slow the progression of the disease,
however, new studies show that
short non-coding RNA molecules
called MicroRNAs have the potential
to predict and track the progression
of ALS in patients, allowing doctors
and researchers to better
understand the disease.
Background
Information
ALS Ice Bucket
Challenge
A popular internet challenge that
raised awareness for ALS and
managed to raise over $115 million to
go towards ALS research just in 2014.
Background Information
Amyotrophic Lateral Sclerosis - also known as ALS or
Lou Gehrig’s disease - is an incurable disorder
characterized by the loss of motor neurons in the
central nervous system over time.

Very few ALS patients survive for over ten years after
diagnosis; most die within 2-4 years due to respiratory
insufficiency and dysfunction of the medulla oblongata
in the brain (controls the heart and lung functions).

To date, there is no cure to ALS.

Current Treatment
There are only two drugs that have been verified by
the US Food and Drug Association (FDA) are Riluzole
and Edaravone. The purpose of both of these drugs is
simply to slow the progression of the disease to be
able to prolong the life of the patient by a few months.
ALS Types and Symptoms
Generalized Symptoms: Slurred or nasal speech and difficulty chewing or swallowing

Dysfunction of Upper Motor Neurons: Dysfunction of Lower Motor Neurons:

(Motor Cortex of the Brain) (Brainstem & Spinal Cord)
● Hyperreflexia ● Generalized weakness
○ Your nervous system involuntary ● Muscle atrophy
overreacts to stimuli. Rapid heartbeat, ○ When your muscles waste away
blood pressure spike, constriction of blood ● Hyporeflexia
vessels.
○ When your reflexes are below normal or
● Extensor plantar response absent
○ Also known as Babinski’s sign, this reflex ● Fasciculations
is when you stroke an object along a
○ A sudden twitch or contraction of the
person’s foot from the heel to the toe, the
muscles under the skin
toes fan out.
● Muscle cramps
● Increased muscle tone
 Initial Symptoms
Many patients experience different symptoms
before they are diagnosed with ALS. Some of
these symptoms include:
● Difficulty holding or using a pen
● Inability or difficulty buttoning up a shirt
● Difficulty lifting a cup of coffee

My uncle’s first sign was the inability to flip a

hamburger on the grill.
What are biomarkers?
The National Institutes of Health Biomarkers Definitions Working Group describe
biomarkers as “a characteristic that is objectively measured and evaluated as an indicator
of normal biological processes, pathogenic processes, or pharmacologic responses to a
therapeutic intervention”

This basically means that biomarkers are markers or indicators in an organism that can be
measured. In this case, biomarkers (MicroRNAs) can be used to track the progression of
ALS in the body.
What are MicroRNAs?
MicroRNAs - Also called miRNAs - are short (about 22 nucleotides) non-coding RNA molecules that
regulate gene expression in an organism.

MicroRNAs are extremely stable and can be measured in many different body fluids including blood,
tears, saliva and cerebrospinal fluid.

miRNAs are also resistant to boiling, pH changes, and repeated freezing and thawing.

Due to these characteristics, miRNAs may

make promising biomarkers to help
researchers develop a test that could aid
doctors to diagnose patients with ALS, but
also to track the progression of the disease in the
body.
Methodology
Four Ways to Profile miRNAs
1. Microarray:

A grid of DNA segments of known sequence

(in this case, specific biomarkers or miRNA)
that is used to test and map DNA fragments,
antibodies, or proteins
Four Ways to Profile miRNAs
2. Quantitative Real-Time Polymerase
Chain Reaction (qRT-PCR)-based array

Like a normal PCR reaction, but it monitors

the amplification of the targeted DNA
sequence during the PCR, not just at the
end.
Four Ways to Profile miRNAs
3. Quantitative nCounter

NanoString nCounter® gene expression

system is a recently developed RNA-based
technology that allows digital quantification
of multiplexed target molecules through the
use of color-coded barcodes.
Four Ways to Profile miRNAs
4. Next-Generation Sequencing (NGS)

Targeted sequencing allows you to

sequence a subset of genes or specific
genomic regions of interest
Classifications of sensibility,
specificity, and throughput:

+++ (very high)

++ (moderate)

+/++ (moderate to low)

+ (low)
Cerebrospinal Fluid (CSF)
The fluid that bathes your central nervous
system, this makes it an ideal source for
identifying biomarkers.

However, the CSF samples require a puncture

to the lumbar portion of the spine. This is an
invasive procedure that cannot be done
multiple times with one patient due to ethical
issues.
Blood
Unlike CSF, getting blood samples from
patients is a non-invasive way to get a
reliable source of biomarkers. Plus
samples can be taken more than once,
giving researchers the ability to attempt
to duplicate their findings.
Muscle Biopsy
This is a procedure in which a small
sample of tissue is removed from the
patient for testing.

Skeletal muscle is also a reliable source

for finding ALS biomarkers. This
however, is also an invasive procedure
that cannot be repeated on the same
patient, meaning that doctors cannot
continually track the progression of the
disease using this source.
Data & Analysis
Table 1
These are the five experiments
that have been done to find
biomarkers using CSF.
Table 2
These next experiments were
done by studying blood,
leukocytes (white blood cells),
or a serum of specific miRNAs.
Table 4
These experiments show
different miRNAs identified
using different muscle tissues
in ALS and healthy control
volunteers.
Concerns
One of the main topics of
my paper was that all of
these experiments have
been done, yet many of
them are not reproducible
and all of the researchers
used different methods. “A common acceptance of certain guidelines,
standard research protocols, and strong
methods of statistical analysis of miRNA will
be important in the future to achieve reliable
biomarkers.”
(pg. 14)
Conclusion
ALS is a mystery to doctors and researchers. There is no
chance of survival, and no reliable treatment. Up until
recent years no one has even understood ALS. Now,
researchers are working to identify specific miRNAs so they
can better diagnose ALS, as well as track the progression of
ALS in the patient. Using these biomarkers
can lead to more understanding of ALS and
perhaps eventually, viable treatment or even
a cure.
Further Studies
Future studies will need to be done to
streamline results and generate more
accurate and non conflicting results.
“Future studies should try to
combine data obtained from
multiple source of sample
(blood, CSF, muscle biopsy) of
the same patient...an extensive
analysis of correlations among
different samples could be
helpful to obtain more
informative data”
(pg. 14)