Background Cluster headache (CH) is a primary headache included in the trigeminal
autonomic cephalalgias (TACs) according to the International Calssification of Headache
Disorder, Third Edition. CH is characterized by a multifaceted and incompletely
understood pathophysiology. The hypothalamus, the trigeminal-vascular complex, and the
trigeminal-autonomic reflex are believed to play a significant role.
Increasing experimental evidence has highlighted the involvement of specific microRNAs
(miRNAs) in the chronic pain and primary headaches, including migraine. miRNAs are small
endogenous noncoding RNAs that are around 22 nucleotides in length. miRNAs operate as
post-transcriptional regulator of gene expression by promoting messenger RNA (mRNA)
degradation or repressing mRNA translation. The regulation process performed by miRNAs is
complex and articulated since an individual miRNA might target hundreds of different
mRNAs, and conversely, each mRNA may be regulated by multiple miRNAs. It has been
estimated that more than 60% of all protein-coding genes are regulated by miRNAs which
consequentially determine the pleiotropic modulation of a wide variety of cellular
processes involving differentiation, development and signaling. miRNAs are involved in
the generation and maintenance of pain and several evidence suggest that specific miRNAs
could play a role in migraine.
In a recent study, we observed an upregulation of gene expression in two miRNAs,
miR-382-5p and miR-34a-5p, in peripheral blood mononuclear cells (PBMCs) in subjects with
chronic migraine (CM). These miRNAs are involved in inflammation modulation and the
release of γ-aminobutyric acid (GABA) compared to individuals with episodic migraine and
healthy subjects (HCs). These findings underscore a correlation between the gene
expression of these miRNAs and the migraine phenotype, as well as its severity.
Recent studies suggest an active role for certain neuropeptides in CH. The Danish group
has indeed demonstrated that the intravenous administration of CGRP, PACAP, or VIP can
induce acute CH attacks in at least 50% of participants when studied in the active phase
of the disease.
As of now, there is no data regarding the potential involvement of miRNAs in CH. This
study aims to provide a detailed exploration of the role of miRNAs in cluster headache,
contributing to our understanding of the pathophysiology of this primary headache
disorder. Furthermore, the results obtained may pave the way for the development of a new
generation of molecules that could be used in the field of CH, such as miRNA agonists
(Agomir) and antagonists (Antagomir).
Aims The primary objective of our current project is to investigate the peripheral
expression of miRNAs implicated in pain modulation (miR-382-5p, miR-34a-5p, and miR-155)
in subjects with episodic CH in the active phase (eCH-act), episodic CH in the remission
phase (eCH-rem), chronic CH (cCH), and healthy control subjects (HC).
Methods
All subjects will perform a single evaluation over time during which they will undergo:
explanation of study procedures and informed consent signing
screening visit
clinical and demographic data collection (based on revision of a headache diary)
fasting venous blood sampling
administration of questionnaires
All subjects will be evaluated in the morning, after night fasting. For subjects affected
by cCH or eCH-act, sampling is expected to be carried out in the inter-critical phase, as
defined by the absence of CH attacks for at least 3 hours in patients with eCH and for at
least 8 hours in patients with cCH. After blood sampling, patients will stay at Center to
complete a set of questionnaires. This will allow the investigator to monitor the
patients for at least 2 hours, to exclude that a blood sampling is performed immediately
before a CH attack.
For patients with eCH, the "active" and "remission" phases will be defined as follows:
Active phase: the phase during which CH attacks have been present for at least 7
days.
Remission phase: characterized by an absence of cluster headache attacks for at
least 15 consecutive days without pharmacological therapy.
miR-382-5p, miR-34a-5p and miRNA-155 gene expression will be evaluated in PBMCs by
real-time reverse transcription (RT-PCR). This assessment will be normalized with U6 (a
type of small nuclear RNA used as housekeeping gene) and expressed as Relative
Quantification (RQ). Plasma levels of CGRP alpha, PACAP and VIP will be measured using
validated commercial ELISA kits. A detailed description of the methods planned for the
biochemical analyses could be found here: https://doi.org/10.1186/s10194-020-01189-0.
The set of clinical questionnaires will include: Migraine Disability Assessment (MIDAS),
Headache Impact Test (HIT-6), Cluster headache Impact Questionnarie (CHIQ).
Sample size calculation and pre-planned statistical analysis The following parameters
were considered for the calculation: Effect Size: 0.5; alpha error: 0.05; beta error:
0.05 (power: 95%); number of groups: 4.
Sample size calculation was conducted using the freeware platform G*Power (ver. 3.1.9.7)
for an ANOVA test with Bonferroni correction applied to the 4 groups. The minimum
suggested sample size is 76 subjects (19 subjects per group). To account for potential
variability, we plan to enroll a total of 100 subjects, with 25 subjects per group.
Parametric or non-parametric correlations will be used to measure the relationship
between biochemical variables and clinical variables. Multivariate analyses will be
performed according to the results of the univariate analysis. Statistical significance
will be set at the 5% level (p<0.05).