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\begin{document}

\title{Circulating miRNAs -- a potential tool to identify severe asthma risk?}



\author[1]{Aref Kyyaly}%
\author[1]{Tilman Sanchez-Elsner}%
\author[2]{He Peijun }%
\author[2]{Collin Sones}%
\author[2]{S Arshad}%
\author[1]{Ramesh Kurukulaaratchy}%
\affil[1]{University of Southampton Division of Clinical and Experimental Sciences}%
\affil[2]{University of Southampton}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\textbf{Circulating miRNAs -- a potential tool to identify severe asthma
risk?}

\textbf{M Aref Kyyaly\textsuperscript{1,2,3},} \textbf{Tilman}

\textbf{Sanchez-Elsner\textsuperscript{2,3}, Peijun
He\textsuperscript{4}, Collin L Sones4, S Hasan
Arshad\textsuperscript{1,2,3}, and Ramesh J
Kurukulaaratchy\textsuperscript{1,2,3}}

\textsuperscript{\textbf{1}}\textbf{David Hide Asthma and Allergy
Research Centre, Isle of Wight, UK. \textsuperscript{2}Clinical and
Experimental Sciences, Faculty of Medicine, University of Southampton,
Southampton, UK. \textsuperscript{3}Respiratory Biomedical Research
Centre, University Hospitals Southampton NHS Foundation Trust,
Southampton,\textsuperscript{4}Optoelectronics Research Centre,
University of Southampton}

Although one of the commonest chronic diseases, asthma lacks a gold
standard diagnostic test. Reliance on a clinical diagnosis alongside the
heterogeneous nature of asthma means that asthma diagnosis is often
delayed to the detriment of the patient. Therefore, new asthma
diagnostic biomarkers are an important research focus. MicroRNAs
(miRNAs) might hold promise in that regard. These small noncoding RNAs
act through RNA-induced silencing complexes to post-transcriptionally
regulate mRNA (messenger RNA). A previous study demonstrated
differential miRNA expression in blood eosinophils from asthmatics
compared with healthy individuals. They clustered asthmatics and healthy
subjects showing that miRNA profiles in eosinophils (especially
miR-185-5p and miR-1246) are potential serum biomarkers for ranking
asthma severity.(1) Another study from our lab demonstrated that
miR-150, miR-152, and miR-375 are up-regulated in severe asthma.(2)

We determined the differential miRNA expression in sera of severe
asthmatic patients and compared it to mild asthma and no asthma to
assess their potential as biomarkers for the diagnosis of asthma and its
discriminative capability for asthma severity.

We investigated the presence of 175 miRNAs in serum samples collected
from 12 adult subjects (4 healthy, 4 mild asthma, and 4 severe asthma)
of our cohort studies (summary features and definitions, Table 1).
Healthy subjects and mild asthma patients were from the Isle of Wight
Birth Cohort (IOWBC) (3), while severe asthma patients were from the
Wessex AsThma CoHort of difficult asthma (WATCH) study (4). We
hypothesized that asthma-specific miRNAs may differentiate asthmatic
from healthy subjects, while other miRNAs may differentiate mild from
severe asthma.

We extracted miRNAs from stored serum using miRNeasy Serum/Plasma Kit
(QIAGEN). After extraction, RNA was reverse-transcribed to cDNA. We then
evaluated miRNA expression by quantitative PCR using Human Serum/Plasma
Focus, miRCURY LNA miRNA Focus PCR Panel kit (QIAGEN) which is designed
for profiling 175 human miRNAs commonly found in serum and plasma.
Results were analysed using QIAGEN web portal GeneGlobe. At least
two-fold change in expression and P [?]0.05 were required to consider the
miRNA of diagnostic value.

Figure 1 shows volcano plots for comparisons of the studied miRNAs in
severe asthmatic, mild asthmatic, and healthy groups. We found that,
miR-193a-5p was significantly increased while miR-181a-5p, miR-146a-5p,
and miR-16-2-3p were significantly decreased in serum from severe
asthmatics compared to healthy subjects. Furthermore, miR-197-3p,
miR-223-3p, miR-151a-3p, miR-191-5p, and miR-28-3p were significantly
increased and miR-451a, miR-16-2-3p, miR-210-3p, miR-133a-3p, miR-660-5p
and miR-144-3p significantly decreased in serum from severe asthmatics
compared to mild asthmatics. There were no significant differences in
expression of the studied miRNAs in serum between healthy subjects and
mild asthma patients.

Our preliminary study demonstrated differential sera miRNA expression in
severe asthma compared to both mild asthma and healthy subjects. We also
found that different miRNAs respectively distinguished severe asthma
from mild asthma than from healthy controls. Such findings emphasise the
distinctive nature of severe asthma and may aid understanding of
mechanistic differences between mild and severe asthma. To our
knowledge, miR-28-3p, miR-16-2-3p, and miR-210-3p have not been
previously reported as differentially expressed miRNAs in asthma while
miR-151a-3p has been inversely correlated with Bronchoalveolar lavage
(BAL) eosinophil percentage in asthma patients.(5) We tested 175 miRNAs
within a standard panel but other miRNAs previously reported in the
literature may differentiate asthmatic patients from healthy
subjects.(1, 2) Furthermore, asthma is a heterogeneous condition so
different miRNAs may be relevant to different phenotypes and endotypes.
Differential miRNA expression may be seen in different tissues too. Our
pilot study shows that miRNA expression may help to distinguish severe
asthma status. Larger scale work studying miRNA expression in different
asthma subgroups, different tissue compartments and using a wider array
miRNAs is therefore warranted.

Table1: Clinical characteristics for subjects from different groups.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
Severe asthma N=4\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
Mild asthma N=4\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
Healthy N=4\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Definition of Asthma Status\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Enrolled in WATCH study and taking BTS steps additional controller
therapies/specialist therapies (6)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Enrolled in IOWBC. Physician diagnosis of asthma at age 18-years plus
either wheeze within the last 12 months or currently taking asthma
medication at BTS steps regular preventer/ initial add on therapy
(6)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Enrolled in IOWBC. No documented history of asthma ever at age
18-years\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Gender (F) \% (N)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
75 (3)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
75 (3)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
100 (4)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Current Smoker \% (N)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
50 (2)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
25 (1)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Never smoked \% (N)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
50 (2)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
25 (1)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
75 (3)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
ICS dose BDP equivalent \selectlanguage{greek}μ\selectlanguage{english}g/day\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
2500 (1000-3000)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
400 (0-800)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
NA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
BMI (Kg/m\textsuperscript{2})*\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
31.8 (22.1-39.6)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
23.9 (20.2-26.4)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
21.7 (19.0-24.6)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Rescue OCS (in last 12 months) (N)*\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
3.7 (1-7)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
On Maintenance OCS\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
No\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
No\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
No\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
On Asthma Biologics \% (N)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0 (0)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
FEV1 (\%)*\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
80.84 (68.73-96.76)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
3.88 (2.97-5.37)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
3.69 (3.54-4.03)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
FVC (\%)*\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
94.63 (82.88-106.72)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
4.68 (3.34-6.96)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
4.15 (3.83-4.45)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Total IgE (KU/L)*\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
1463.7 (19.6-2907.9)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
119.4 (5.6-346.0)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
210 (36-384)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Atopy \% (N)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
50 (2)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
75 (3)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
50 (2)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Data are presented as \%, mean. BMI: body mass index; ICS: inhaled
corticosteroids; BDP: beclometasone dipropionate; OCS: oral
corticosteroids; FEV1: forced expiratory volume in 1 s; FVC: Forced
vital capacity. *Mean (range). NA = Not applicable WATCH= Wessex AsThma
CoHort. IOWBC= Isle of Wight Birth Cohort. BTS=British Thoracic Society.

References:

\begin{enumerate}
\tightlist
\item
  Rodrigo-Mu\selectlanguage{ngerman}ñoz JM, Cañas JA, Sastre B, Rego N, Greif G, Rial M, Mínguez
  P, Mahíllo-Fernández I, Fernández-Nieto M, Mora I, Barranco P, Quirce
  S, Sastre J, Del Pozo V. Asthma diagnosis using integrated analysis of
  eosinophil microRNAs. Allergy. 2019 Mar;74(3):507-517. doi:
  10.1111/all.13570. Epub 2018 Oct 11. PMID: 30040124.
\item
  Rupani H, Martinez-Nunez RT, Dennison P, Lau LC, Jayasekera N,
  Havelock T, Francisco-Garcia AS, Grainge C, Howarth PH, Sanchez-Elsner
  T. Toll-like Receptor 7 Is Reduced in Severe Asthma and Linked to an
  Altered MicroRNA Profile. Am J Respir Crit Care Med. 2016 Jul
  1;194(1):26-37. doi: 10.1164/rccm.201502-0280OC. PMID: 26815632;
  PMCID: PMC4960627.
\item
  Arshad SH, Holloway JW, Karmaus W, Zhang H, Ewart S, Mansfield L,
  Matthews S, Hodgekiss C, Roberts G, Kurukulaaratchy R. Cohort Profile:
  The Isle Of Wight Whole Population Birth Cohort (IOWBC). Int J
  Epidemiol. 2018 Aug 1;47(4):1043-1044i. doi: 10.1093/ije/dyy023. PMID:
  29547889; PMCID: PMC6124620.
\item
  Azim A, Mistry H, Freeman A, Barber C, Newell C, Gove K, Thirlwall Y,
  Harvey M, Bentley K, Knight D, Long K, Mitchell F, Cheng Y,
  Varkonyi-Sepp J, Grabau W, Dennison P, Haitchi HM, Arshad SH,
  Djukanovic R, Wilkinson T, Howarth P, Kurukulaaratchy RJ. Protocol for
  the Wessex AsThma CoHort of difficult asthma (WATCH): a pragmatic
  real-life longitudinal study of difficult asthma in the clinic. BMC
  Pulm Med. 2019 May 24;19(1):99. doi: 10.1186/s12890-019-0862-2. PMID:
  31126281; PMCID: PMC6534885.
\item
  Francisco-Garcia AS, Garrido-Martín EM, Rupani H, Lau LCK,
  Martinez-Nunez RT, Howarth PH, Sanchez-Elsner T. Small RNA Species and
  microRNA Profiles are Altered in Severe Asthma Nanovesicles from
  Broncho Alveolar Lavage and Associate with Impaired Lung Function and
  Inflammation. Noncoding RNA. 2019 Nov 2;5(4):51. doi:
  10.3390/ncrna5040051. PMID: 31684064; PMCID: PMC6958500.
\item
  British guideline for the management of asthma. A national clinical
  guideline. British Thoracic Society/NHS Scotland. 2019.
\end{enumerate}

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