Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT03938909 |
| Other study ID # |
CGM-03 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
March 1, 2019 |
| Est. completion date |
January 1, 2025 |
Study information
| Verified date |
April 2022 |
| Source |
Neuromed IRCCS |
| Contact |
Stefano Gambardella, PhD |
| Phone |
+39 0865 915 209 |
| Email |
stefano.gambardella[@]neuromed.it |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational [Patient Registry]
|
Clinical Trial Summary
There is a long history of research into body fluid biomarkers in neurodegenerative and
neuroinflammatory diseases. However, only a few biomarkers in cerebrospinal fluid (CSF) are
being used in clinical practice. One of the most critical factors in biomarker research is
the inadequate linkage of biological samples with data from medical records, environmental
exposure, lifestyle information and other medically relevant information. In this context the
biobanks are an invaluable resource for medical research and, in particular, for the
identification of biomarkers. This project aims to enstablish a biobank for Multiple
Sclerosis that allow to collect periodically, at each follow up, clinical data, tissues such
as blood and cerebrospinal fluid and DNA, RNA, proteins, from patients afferent at the Centre
for the Study and Cure of Multiple Sclerosis in Neurological Institute "Neuromed", Pozzilli,
Isernia. The samples stored in this biobank are examined by quantization of a potential
innovative biomarker focused on the formation of circulating mitochondrial DNA. Fragments of
mitochondrial DNA (mtDNA) are released outside the cell and they appear to persist in
extracellular fluids as circulating, cell-free, mtDNA (ccf-mtDNA).
This occurs during acute inflammation, which anticipates the neurodegenerative process. Thus,
an increase in inflammatory cells in the affected regions is expected to add on mtDNA release
into the CSF. Thus, ccf-mtDNA may represent a powerful biomarker for disease screening and
prognosis at early stage, although its biological role may extend to generating the
neurobiology of disease.
Aims:
1. Identify a technique that allows to isolate, the mitochondrial DNA circulating from
different biological tissues (Droplet Digital PCR, Real Time PCR).
2. Use different technologies to quantify the presence of circulating mitochondrial DNA
3. Use circulating mitochondrial DNA as a biomarker of neurodegenerative and / or
neuroinflammatory pathologies.
It is essential to understand the tissue specific origin of circulating mtDNA for both
diagnostic and therapeutic considerations. . We believe that our current knowledge on cell
free circulating mtDNA is in a rather exploratory phase with a potential for the future to
rewrite the pathology of the leading causes of morbidity and mortality such as inflammatory
conditions, autoimmune disorders, cancer, heart disease, stroke and injury.
Description:
To date, the specific causes of multiple sclerosis (MS) remain uncertain, and in its
pathogenesis an interaction between environmental and genetic factors has been implicated
leading to inflammation, demyelination and neurodegeneration of the central nervous system
(CNS). Epidemiological studies conducted in ethnic groups of families, twins, half sibs and
conjugate pairs support a genetic component to this process. The risk for monozygotic twins
is 300-times, and for first-degree relatives 20-50-times higher than for an individual in the
general population of Northern-European origin with a prevalence rate of 0.1. The
transmission patterns observed are not compatible with an autosomal dominant, recessive or
X-linked inheritance. MS is a complex trait disorder, defined by several genes, each exerting
small effect, and in an interaction with the environment. Phenotypic expressions of MS
suggest the involvement of complex mechanisms with features of autoimmunity and
neurodegeneration. The currently approved disease modifying drugs are mainly targeted towards
the inflammatory components, but exert a limited effect on neurodegeneration in MS. The
cognitive impairment can be an early feature of the demyelinating disease process, and in a
few cases dementia has been documented in the absence of severe neurological signs. There are
some correlation between disease subtype and cognitive impairment: in fact, it is well known
that cognitive impairment occurs more frequently and is more severe in patients with
progressive rather than in relapsing-remitting MS. Impairment of cognitive domains such as
memory, mental processing speed attention and executive function can occur from the early
stage of the diseases and tend to worsen over time. Moreover, the underlying
pathophysiological mechanisms of the cognitive impairment and neuropsychiatric disorders
observed in MS are not fully understood. White matter abnormalities alone cannot fully
explain the extent of clinical symptoms in MS, including cognitive impairment. Furthermore,
several MRI techniques have shown the involvement of gray matter in MS and the association
between gray matter damage, physical disability and cognitive impairment. Therefore,
biomarkers that reliably capture the different aspects of disease heterogeneity are needed,
and might help to better understand MS disease aetiopathogenesis, diagnosis, and prognosis,
to predict response outcome to treatments, and to develop new treatments.
In particular, there is increasing effort to develop molecular diagnostic markers that meet
requirements like easy accessibility e.g., from blood, high specificity and sensitivity, low
costs and applicability by laboratories with standard equipment. Several blood, plasma, or
serum MS biomarkers have been proposed to meet these criteria. In order of this the
circulating markers are represented, in addition to the classic serum markers, also by the
cells and by free circulating nucleic acids (DNA, RNA). In recent years, among the
circulating nucleic acids, a possible role of mitochondrial DNA has emerged as a biomarker in
the diagnosis of numerous pathologies.
In humans, mtDNA is significantly smaller when compared with nuclear DNA (16.569bp vs. 3.2
billion bp), and it possesses only 37 genes, among which 13 encode proteins belonging to the
respiratory electron transport chain. Unlike nuclear DNA, mtDNA is devoid of protective
histones and sophisticated DNA repair mechanisms, which makes it vulnerable to genotoxic
stimuli including oxidative stress. In fact, high levels of reactive oxygen species (ROS) are
generated around mtDNA during oxidative phosphorylation occurring in mitochondria. Such an
oxidative environment contributes to a high susceptibility of mtDNA to mutagenesis. In fact,
mtDNA possesses roughly a 10- to 200-fold higher rate of mutagenesis than nuclear DNA under a
comparable oxidative stress environment. This may be detrimental for those
high-energy-demanding and post-mitotic cells including neurons and myocytes, which are mostly
sensitive to altered respiratory chain activity and ROS-mediated damage yielded by mtDNA
changes. Such a specific vulnerability of mtDNA determines the occurrence of a detectable
amount of mitochondrial DNA fragments, which are released into the bloodstream as
circulating, cell-free fragments (ccf-mtDNA). These correspond to double-stranded DNA
molecules, which are biologically fragmented into both short (lower than 1 Kb) and long (up
to 21 kb) segments. The high rate of mtDNA fragmentation is key in generating ccf-mtDNA,
though it remains unclear whether mtDNA is released due to a disruption of the plasma
membrane or it is actively extruded from the cell. For instance, oxidative stress or other
stimuli can damage cell integrity, while producing apoptosis or necrosis, which in turn lead
to mtDNA extrusion from the cell or release into the blood. Nonetheless, even in baseline
conditions when the plasma membrane is intact, fragments of mutated mtDNA could be
compartmentalized within cytosolic organelles and then released extracellularly. This latter
mechanism would guarantee the preservation of mitochondrial function by removing
dysfunctional mutated DNA fragments. This is supported by recent work from C. elegans
neurons, which expel dysfunctional mitochondria when exposed to neurotoxic stress.
Nonetheless, the biological role of ccf-mtDNA and its fragments is still controversial and it
needs to be fully understood. In fact, DNA fragments may act as toxic molecules, which in
turn impair mitochondrial function and cell membrane, and could also act on cell integrity
and tissue repair. This is largely bound to the established, yet double faceted, involvement
of mtDNA in innate immunity and inflammation. In fact, similar to bacterial DNA, mtDNA
possesses non-methylated CpG sites, which once released in either cytosol or extracellular
space behave as damage-associated molecular patterns (DAMPs) to activate innate immunity and
inflammation. This occurs via specific biochemical cascades involving the binding of mtDNA to
Toll-like receptor 9 (TLR9) and subsequent activation of the stimulator of interferon genes
(STING) pathway. These are key in generating inflammatory responses including antimicrobial
immunity and neuro-immunological disorders. In fact, DAMPs accumulation activates resident
macrophages and fosters tissue infiltration by leukocytes. As for most molecules involved in
the immune response, the bulk of evidence concerning the measurement of ccf-mtDNA and its
role in physiology and disease stems from studies carried out outside the CNS. In fact,
ccf-mtDNA has been analysed in various clinical conditions like neoplasia, trauma,
infections, stroke and cardiovascular diseases, where it has been tested as diagnostic and
predictive biomarker. Only recently, mtDNA started being evaluated in neurological disorders.
In line with the higher resistance of mtDNA to nuclease-dependent degradation compared with
nDNA, mtDNA persists as ccf-mtDNA within extracellular fluids including the CSF.
CNS disorders featuring a strong inflammatory response are characterized by elevated plasma
mtDNA level. In fact, elevated CSF ccf-mtDNA occur in relapsing-remitting (RRMS) and PMS.
RRMS is characterized by an acute inflammatory response, which precedes neurodegeneration.
Thus, the increase in ccf-mtDNA observed in RRMS is a direct consequence of increased
activation of inflammatory cells. These cells release mtDNA in addition to nDNA, into the CSF.
A persistent inflammatory reaction may recruit circulating immune cells while triggering a
systemic response through the activation of mtDNA-induced inflammatory pathways. In this way,
a vicious circle occurs where inflammatory cytokines and ROS may induce further damage to
mitochondria and mtDNA. In this scenario, elevated ccf-mtDNA concentration in MS may reflect
early, active inflammatory activity, which eventually culminates in mitochondrial damage,
neural loss and brain atrophy. In this condition, the measurement of ccf-mtDNA concentration
configures as a potential biomarker for acute inflammatory stress. Whether this phenomenon is
specific for MS or it rather reflects a generic neuro-inflammation still needs to be
investigated. Since mitochondrial damage occurs in active MS lesions, mtDNA in the CSF could
reflect its role as a DAMP. Considering mtDNA as a DAMP in MS, may explain the "inside-out
theory" which suggests that inflammation is secondary to a primary intrinsic process within
neurons or other cells such as oligodendrocytes. This neuro-immune concept consists in the
formation of intracellular compounds, which trigger biochemical cascades leading to immunity
activation (inflammasome) which once released from the cell recruit in turn a focal immune
response. In this scenario, the "inside" mtDNA fragment would be the inflammatory stimulus,
which clusters the intracellular cascade leading to a molecular complex, which triggers the
immune response. Once such a complex is exposed "out" of the cell, immunity is strongly
activated.
Thus, ccf-mtDNA may be a potential biomarker of cell death and non-specific tissue injury,
and in the near future, it is supposed to become an innovative diagnostic tool in early stage
screening and prognosis of several disorders.
