Received: 17 October 2018 | Revised: 10 February 2019 | Accepted: 14 February 2019

DOI: 10.1002/jcp.28482

MINI‐REVIEW

Identification of biomarkers and genetic approaches toward

chronic obstructive pulmonary disease

Ridhima Wadhwa1 | Taru Aggarwal2* | Vamshikrishna Malyla3,12 | Nitesh Kumar4 |

Gaurav Gupta5 | Dinesh Kumar Chellappan6 | Harish Dureja7 | Meenu Mehta8 |
Saurabh Satija8 | Monica Gulati8 | Pawan Kumar Maurya9 | Trudi Collet10 |
Philip Michael Hansbro11,12,13 | Kamal Dua3,11,12
1
Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India
2
Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India
3
Discipline of Pharmacy, Graduate School of Health, University of Technology Sydney, New South Wales, Australia
4
Amity Institute for Advanced Research & Studies (M&D), Amity University, Noida, Uttar Pradesh, India
5
School of Pharmaceutical Sciences, Jaipur National University, Jagatpura, Jaipur, Rajasthan, India
6
Department of Life Sciences, School of Pharmacy, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia
7
Department of Pharmaceutical Sciences, Maharishi Dayanand University, Rohtak, Haryana, India
8
School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India
9
Department of Biochemistry, Central University of Haryana, Mahendergarh, Haryana, India
10
Innovative Medicines Group, Institute of Health & Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
11
Priority Research Centre for Healthy Lungs, University of Newcastle & Hunter Medical Research Institute, Newcastle, New South Wales, Australia
12
Centre for Inflammation, Centenary Institute, Sydney, New South Wales, Australia
13
School of Life Sciences, University of Technology Sydney, Sydney, New South Wales, Australia

Correspondence
Ridhima Wadhwa, Faculty of Life
Sciences and Biotechnology, South Asian
University, Akbar Bhawan, Chanakya-
puri, New Delhi 110021, India.
Email: rw4565@gmail.com;
Kamal Dua, Discipline of Pharmacy,
Graduate School of Health, University of
Technology Sydney, P.O. Box: 123
Broadway, New South Wales 2007,
Australia.
Email: Kamal.Dua@uts.edu.au
Funding information
National Health Medical Research Council,
NHMRC, Grant/Award Number: 1079187
Abstract
Chronic obstructive pulmonary disease accounts as the leading cause of mortality
worldwide prominently affected by genetic and environmental factors. The disease is
characterized by persistent coughing, breathlessness airways inflammation followed
by a decrease in forced expiratory volume1 and exacerbations, which affect the
quality of life. Determination of genetic, epigenetic, and oxidant biomarkers to
evaluate the progression of disease has proved complicated and challenging.
Approaches including exome sequencing, genome‐wide association studies, linkage
studies, and inheritance and segregation studies played a crucial role in the
identification of genes, their pathways and variation in genes. This review highlights
multiple approaches for biomarker and gene identification, which can be used for
differential diagnosis along with the genome editing tools to study genes associated
with the development of disease and models their function. Further, we have
discussed the approaches to rectify the abnormal gene functioning of respiratory
tissues and various novel gene editing techniques like Zinc finger nucleases (ZFN),
transcription activator‐like effector nucleases (TALEN), and clustered regulatory
interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9).

*Taru Aggarwal contributed equally.

J Cell Physiol. 2019;1–21. wileyonlinelibrary.com/journal/jcp © 2019 Wiley Periodicals, Inc. | 1

2 |
KEYWORDS
biomarkers, cellular therapy, chronic obstructive pulmonary disease, CRISPR/Cas‐9, genome
editing, oxidative stress
1 | INTRODUCTION
According to the World Health Organization (WHO), chronic
obstructive pulmonary disease (COPD) is defined by chronic
obstruction of airways interfering with normal lung breathing
which is not completely reversible. It is characterized by
emphysema, airway fibrosis, and chronic bronchitis, which ob-
struct the normal physiological breathing due to multiple factors
like genetic, environmental, and cigarette smoking (World Health
Organization, 2019). Global obstruction for chronic lung disease
(GOLD) report 2019, defines COPD as prevalent, preventable, and
treatable disease distinguished by persistent respiratory symp-
toms and airflow limitation because of airways/alveolar anomaly
due to noxious particles and gases exposure (Global Initiative for
Chronic Obstructive Lung Disease, 2019).The disease is associated
with dyspnea, cough or sputum production obstruction of airflow,
inflammation in the lungs, emphysema, and tissue tear down
(Global Initiative for Chronic Obstructive Lung Disease, 2019;
Mapel, Dalal, Johnson, Becker, & Hunter, 2015). Worldwide, COPD
is the fourth leading cause of mortality and by 2020, it is expected
to be the third major cause of death (Global Initiative for Chronic
Obstructive Lung Disease, 2019). According to the WHO report in
2016, 251 million cases of COPD along with 3.15 million deaths
per year have been reported (Mathers & Loncar, 2006). In low and
middle income countries the burden of disease is much higher due
to environmental pollution and smoking, which have significant
association in progressive symptoms and functional impairment of
airways due to accumulation of oxidative and carbonyl stress
(Banerjee, 2014; Lopez et al., 2006; McGuinness & Sapey, 2017;
World Health Organization, 2017).
COPD is not only caused by environmental factors but also the
genetic predisposition of an individual (Kennedy, Chambers, Du, &
Dimich‐Ward, 2007). It is systematic inflammation predominantly
marked in lung parenchyma and peripheral airways (Barnes, 2016).
The chronic obstruction of lung airways is majorly due to
disintegration and fibrosis of airways owing to lung parenchyma
rigidity which traps air in an irreversible manner (Barnes, 2014).
Only, the cholinergic contractions in the small airways are
reversible in nature. Hypersecretion of mucus is also responsible
for the obstruction of airways, thereafter occupying the lumen
space rendering ciliary dysfunction causing accelerated seizure of
lung function (McDonough et al., 2011). The inflammatory mechan-
ism is found similar to that of smokers but is amplified by airways
obstruction in patients with COPD. In such conditions, airway
lumen recruits a large number of macrophages and neutrophils
while airway walls & parenchyma renders increased levels of
macrophages, T lymphocytes, and B lymphocytes (Brusselle, Joos, &
WADHWA ET AL.
Bracke, 2011; Ezzati Givi, A Redegeld, Folkerts, & Mortaz, 2012).
Thus, the disease is characterized by both innate and adaptive
immune response. Spirometry based diagnoses are used for
identification of COPD that gives permanent decline in forced
expiratory volume (FEV) per second along with the ratio of FEV1 to
forced vital capacity (FVC) that is FEV1/ FVC. The FEV1 and FVC
give a relationship to dose‐response relationship to exposure to
smoke. Depending on post‐bronchodilator FEV1 GOLD classifies
COPD into 4 stages namely: GOLD1 is mild COPD with FEV1 > =
80%, GOLD2 is moderate COPD with 50% = < FEV1 < 80%, GOLD3
is severe COPD with 30% = < FEV1 < 50% and GOLD4 is very severe
COPD with FEV1 < 30% (Mapel et al., 2015). However, this indicates
that the environmental factors and cigarette smoke and genetic
constituents are responsible to cause COPD. The genetic variations
of α‐1 anti‐trypsin (AAT) gene serpin peptidase inhibitor, clade A,
member 1 (SERPINA1) have been regarded to be the major
contributing factor for disease pathogenesis but AAT deficiency
has been interpreted up to 2% of all the causes of COPD over 45
years (Berndt, Leme, & Shapiro, 2012). Therefore, it is essential to
decipher the genetic and environmental causes of COPD as it
involves both epigenetic factors along with somatic level mutations
(Tzortzaki et al., 2012; Yao & Rahman, 2012).
This review intent to emphasize the role of genes and biomarkers
involved in COPD along with the current approaches used to treat
the disease and how the genome editing approach can be used as
therapeutic for treating the disease.
2 | AIRWA YS B IOMA RK ERS O F
O XI D A T I V E S T R E S S I N CO P D
The environmental risk factors including air pollution, dust, and
fumes, smoke from biomass combustion increases the probability of
disease in nonsmokers (Mannino & Buist, 2007). While cigarette
smokers are highly susceptible to disease as smoke from a cigarette
contains 1017 oxidants per puff (Pryor & Stone, 1993). This leads to
direct exposure of lung epithelial cells to inflammation in neutrophils,
macrophages, and lymphocytes. In response to the inflammation
caused, antiproteases and antioxidant factors are released to
counterbalance the damage by reactive oxygen species (ROS) and
proteolytic enzymes (Larsson, 2007). ROS includes superoxide anion
(O2−), hydroxyl radical (OH−) along with other derivatives of oxygen
with unpaired electrons like hydrogen peroxide (H2O2). ROS
formation is a continuous process as a result of metabolic activities
in the cell. However, a great amount of ROS is produced by
neutrophils and macrophages to encounter the inhaled particles
(Rahman, Biswas, & Kode, 2006). The oxidative stress produced
WADHWA ET AL. | 3

F I G U R E 1 Source and mechanism of oxidative stress in chronic obstructive pulmonary disease. Oxidative stress (OS) can be due to the
endogenous and exogenous source. Oxidative stress is a major factor leading to chronic obstructive pulmonary disease (COPD). The exogenous
sources of OS are air pollutants (NO2, SO2), cigarette smoke and infectious agents including bacteria, fungus, and viruses. While the endogenous
sources of OS are inflammatory cells and mitochondrial dysfunction. In case of COPD, oxidative stress stimulates mucus hypersecretion,
inflammation, oxidation of extracellular matrix (ECM), cellular leakage, upregulation of factors including chemokines, tumor necrosis factor–ß (TNF‐ß),
nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB) therefore leading to cell senescence. Furthermore, disintegrates the
homeostasis within the airways by elevated secretion of proteases, elastase, cytokines, perforins, and granozymes, which causes the production of
carbonyl modified autoantibodies, cytotoxicity, and decline in phagocytosis activity [Color figure can be viewed at wileyonlinelibrary.com]

damages lipids, DNA, and protein as represented in Figure 1. ROS are
highly reactive in nature so they have a short half‐life, their level
can be directly estimated in the body and tissue fluids by
measuring target products of oxidation such as oxidized proteins,
lipid peroxidation, antioxidants, and so forth (Dalle‐Donne, Rossi,
Colombo, Giustarini, & Milzani, 2006).
2.1 | Lipid peroxidation
Oxidative stress leads to lipid peroxidation causing oxidative damage
(Niki, 2009). Oxidation of lipids occurs readily by nonenzymatic and
enzymatic oxidants. Polyunsaturated fatty acids are prone to free
radical oxygen producing aldehydes such as lipid hydroperoxides as
the product (Iuliano et al., 2003). Malondialdehyde (MDA) and
thiobarbituric acid reactive substances (TBARS) are the most
commonly used biomarkers for lipid peroxidation (Iuliano et al.,
2003). In the TBARS method, MDA reacts with thiobarbituric acid
(TBA) at acidic pH a stable chromophore with maximum absorbance
at 586 nm (Jammes, Steinberg, Ba, Delliaux, & Brégeon, 2008). Also,
the product of the reaction can be examined by using high
performance liquid chromatography (HPLC). Spectroscopic and
HPLC analysis infers that the level of lipid peroxidation is high in
COPD cases compared to healthy controls (Sünnetçioğlu, Alp,
Sertogullarından, Balaharoglu, & Gunbatar, 2015).
2.2 | Protein oxidation products
The most abundant oxidative damage product of proteins is protein
carbonylation (Berlett & Stadtman, 1997). These carbonyl derivatives
are formed by oxidation of lysine, arginine, threonine, and proline
residue side chains. Proteins can react directly to ROS to produce
peptide fragments with a carbonyl group. The common method of
detection of carbonylation of proteins is with 2,4‐dinitrophenylhy-
drazine (DNPH) which forms dinitrophenylhydrazone (DNP; Levine,
2002). DNP can be estimated spectroscopically at 370 nm or by
immunochemistry with a specific antigen (DNP)‐antibody reaction.
Studies have identified an increase in protein carbonyl level in plasma
of patients with COPD as compared to healthy control. The level of
protein carbonyl increases with disease progression (Santos
et al., 2004).
2.3 | Reactive oxygen species
Several authors have studied the level of O2− in ROS production.
Chemiluminescence‐based method of estimation of O2− has been
used to identify the diseased state. An enzymatic method involving
superoxide dismutase inhibitable reduction of cytochrome c‐ and
nitro‐blue tetrazoliun for reduction of O2− to form diformazan,
which can be measured spectroscopically at 550 nm (Hanta,
4 |
Kocabas, Canacankatan, Kuleci, & Seydaoglu, 2006; Nadeem, Raj,
& Chhabra, 2005).
2.4 | Oxidatively damaged DNA
Oxidation of DNA can be studied by a single cell gel electrophoresis
or comet assay that allows the visualization of DNA breaks. DNA
breaks move out of supercoiled DNA loops to form a comet‐like tail.
The DNA tail gives an estimate of breaks in DNA. This assay is
reliable for the detection of COPD as a significant increase in DNA
damage is observed in COPD compared with the healthy individual
(ben Anes et al., 2014; Lin, Wu, Chen, Hsieh, & Yeh, 2010).
2.5 | Uric acid
Uric acid is an essential antioxidant for the protection of lipoproteins
from oxidation as it acts scavenger of hydroxyl and oxygen free
radicals. The levels can be estimated by an enzymatic method using
spectroscopy and HPLC, which suggests a significant decrease of uric
acid in COPD (Aggarwal, Wadhwa, Rohil, & Maurya, 2018; Nicks,
O'Brien, & Bowler, 2011).
2.6 | Antioxidant nutrients
Plasma levels for antioxidants like vitamin A, C, E, and carotenes can be
estimated spectrophotometric methods. A significant decrease in
vitamin A, C, and E levels have been found in case of COPD
exacerbation, these levels can be restored after exacerbation treatment
(ben Anes et al., 2014; Woźniak, Górecki, Szpinda, Mila‐Kierzenkowska,
& Woźniak, 2013). Along with, a significant decrease in α and ß
carotenes is also found in patients with COPD in comparison to healthy
controls. Trace elements are also an important component to maintain
oxidant‐antioxidant balance. The composition can be studied by X‐ray
emission and mass spectroscopy. Studies reveal that plasma levels of
zinc (Zn), selenium (Se), and potassium (K) are reduced while an increase
in iron (Fe), calcium (Ca), copper (Cu), and rubidium (Rb) has been
observed (Santos et al., 2004).
2.7 | Protein and nonprotein thiols
Thiols are organic compounds containing sulfide group (−SH) which
are important for maintaining the redox balance. Intracellular thiols
like glutathione (GSH) maintain a highly reduced niche in the cell and
the extracellular thiols maintain antioxidant defense system (Turell,
Radi, & Alvarez, 2013). Human serum albumin has highest –SH in a
plasma comprising of 80% of the reduced thiol groups and is essential
in free radical oxygen and nitrogen scavenging (Quinlan, Martin, &
Evans, 2005). Thiols are estimated by Ellman's reagent where free
thiols are reduced to form 5‐thionitrobenzoic acid, which is spectro-
scopically determined at 412 nm (Turell et al., 2013). A significant
reduction in protein and non‐ protein thiols have been observed in all
stages of COPD in comparison to healthy controls. The total
WADHWA ET AL.
GSH/GSSH ratio significantly decreases during COPD exacerbation
in comparison to stable COPD (ben Anes et al., 2014).
2.8 | Total antioxidant capacity
Most commonly the ferric reducing ability of plasma (FRAP) and the
trolox equivalent antioxidant capacity (TEAC) assay have been used
to determine the total antioxidant capacity in the biological samples.
The principle of the FRAP assay is the reduction of ferric
tripyridyltriazine (Fe3 + ‐TPTZ) complex to the ferrous tripyridyltria-
zine (Fe2 + ‐TPTZ) with the formation of blue color, observed at
593 nm. The TEAC assay works by inhibiting antioxidants of the
absorbance of the radical cation of 2, 2‐azinobis (3‐ethylbenzothiazo-
line 6‐sulfonate; ABTS), which has maximal absorption at 660, 734,
and 820 nm. ABTS radical cation is formed by reaction of ABTS with
peroxidase metmyoglobin and H2O2 and on the addition of plasma,
the color change reaction is avoided by suppressed antioxidant
activity. In COPD, both the assays infer a significant decrease in the
total antioxidant capacity (ben Anes et al., 2014).
2.9 | Enzymatic antioxidant activity
Antioxidant enzyme activity includes peroxidase (GSH‐Px), glu-
tathione‐S‐transferase (GST), paraoxonase 1 (PON1), and ceruloplas-
min ferroxidase have been studied in patients with COPD. Refer
Table 1 for a detailed list of antioxidant activity in COPD.
3 | TH E G ENE TIC A PPROAC H
TOWARDS C OPD
COPD is a complex disease, which is caused due to environmental
influence or cigarette smoke, but the relation between genes
interacting with the environment is partially known. Multiple factors
or genes contribute to COPD development where few alterations can
be due to epigenetic modification and others can be due to the
genetic predisposition. The disease progression or pathophysiology is
difficult to study as it is complex to identify genes which are
epigenetically modified and predisposed genetically. Therefore, to
identify predisposed genes to epigenetic modifications several
studies are carried out among various population groups which
include family and segregation studies, linkage studies, candidate
gene association studies, meta‐analysis studies, genome wide analysis
studies, and whole genome and exome sequencing. With the help of
developing technology for diagnosis such as computer tomography
(Kim et al., 2009) or the spirometry, these genotypes can be
correlated phenotypic changes in a precise manner as referred in
Table S1 which summarize the reported and mapped genes
associated with COPD phenotype (DeMeo et al., 2009).
With the help of high‐throughput techniques such as whole genome
sequencing, proteomic studies can correlate disease phenotypes to
genotype. These studies help to identify the proteins which interact in the
biological system (Von Mering et al., 2006). Figure 2 explains the
 WADHWA
ET AL.

T A B L E 1 Antioxidant activity involved in COPD

Enzymatic antioxidant COPD

S.No activity Mode of estimation Mode of action condition Reference
1. Superoxide ELISA, Mc Cord, and Fridovich assay Dismutation of the O2 to molecular oxygen and H2O2. Decline in Quinlan et al., 2005
dismutase (SOD) Xanthine and xanthine oxidase are used to generate O2 and levels
2‐(4‐ iodophenyl)‐3‐(4‐nitrophenol)‐5‐phenyltetrazolium chloride
which reacts with O2 to form a red formazan dye
2. Catalase Spectrophotometry (240 nm), ELISA Detoxification of H2O2 into molecular oxygen and water Not significant ben Anes et al., 2014; Quinlan
et al., 2005
3. GSHPx Oxidation of NADPH Reduction of organic peroxides into alcohols or H2O2 into water Decline in Vibhuti, Arif, Deepak, Singh,
Oxidation of nicotinamide adenine dinucleotide phosphate levels and Pasha (2007)
(NADPH), a coenzyme in the reaction catalyzed by glutathione
reductase that reduces GSSG formed during the activities
catalized by GSHPx
4. GST 1‐Chloro‐ 2,4 dinitrobenzene asa substrate, Inactivates reactive electrophiles by conjugating with GSH Decline in Lakhdar et al. (2011)
spectrophotometry at 340 nm levels
5. PON1 Two‐substrate activity (paraoxon/diazoxon) Hydrolysis of paraoxon protects against the toxicity of some Decline in Isik, Isik, Ceylan, and
method organophosphates and contributes to the antioxidant levels Calik (2005)
protection conferred by HDL on low‐density lipoprotein
oxidation
6. Ceruloplasmin Spectrophotometry (376 nm), Oxidize ferrous ion to ferric ion, complexing with a Elevated levels Healy and Tipton (2007);
ferroxidase Immunoelectrophotometry chromogen Milevoj Kopčinović
et al. (2016)
Abbreviations: COPD: chronic obstructive pulmonary disease; ELISA: enzyme‐linked immunosorbent assay; GST: glutathione‐S‐transferase; PON1: paraoxonase 1
|
5
6 | WADHWA ET AL.

F I G U R E 2 Protein‐protein interaction network in COPD. Genes involved in COPD (as mentioned in Supplementary Table 2) are used to plot
the protein‐protein interaction network using STRING v10: protein‐protein interaction networks, integrated over the tree of life. Szklarczyk D,
Franceschini A, Wyder S, Forslund K, Heller D, Huerta‐Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M, Bork P, Jensen LJ, von
Mering C. The network created contains 49 nodes, 57 edges, with a PPI enrichment p‐value: < 1.0e‐16 [Color figure can be viewed at
wileyonlinelibrary.com]

interaction of various proteins encoded by the crucial gene involved in 3.1 | Genetic predisposition in COPD
COPD. The interaction network represents protein–protein association
COPD aggregates within families, resulting in genetic predisposition
for both epigenetic and predisposed genes based on experiment,
of several genes as evident from Table 3.
predicted expression, coexpression, text mining, and protein homology
One of the majorly discussed genes in this category is IREB2. IREB2
(Table S2: Represents gene name associated with protein). These
is located on 15q25.1, which encodes for protein iron responsive
reported genes can be mapped for airways measurement and COPD
element binding protein 2, a RNA binding protein involved in iron
diagnosis (Table 2).
metabolism inside the human cell (Rouault, 2006). In COPD situations
Genes involved in COPD (as mentioned in Table S2) are used to plot
such as hypoxemia can arise any time; whereas IREB2 is known as iron
the protein‐protein interaction network using STRING v10: protein–
binding protein hence it remains in the active state in lower oxygen
protein interaction networks, integrated over the tree of life. Szklarczyk
conditions (Rouault, 2006). In COPD the expression of IREB in family
D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta‐Cepas J,
cohorts was found to be differentially expressed, according to DeMeo
Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M, Bork P, Jensen LJ,
et al (2009) five IREB2 SNPs (rs1964678, rs2656069, rs12593229,
von Mering C. The network created contains 49 nodes, 57 edges, with a
rs965604, rs13180) have been significantly associated with COPD. Risk
PPI enrichment p < 1.0e−16.
T A B L E 2 Pathogenesis of COPD due to genetic predisposition

Gene Study Chromosomal

WADHWA

S.No symbol Gene name Protein name Sample analyzed population location Mechanism genes involved Reference
ET AL.

1. MMP12 Matrix Macrophage Blood 28 11q22.2 Hydrolysis of soluble and insoluble elastin. Aggarwal
metalloproteinase 12 metalloelastase Specific cleavages are also produced at et al. (2018)
14‐Ala‐|‐Leu‐15 and 16‐Tyr‐|‐Leu‐17 in
the B chain of insulin.
2. FAM13A Family with sequence Fam13a protein Lung tissue sample 5 4q22.1 COPD susceptibility by promoting β‐ Jiang
similarity 13, human + 5 catenin degradation et al. (2016)
member A mice
3. IREB2 Iron‐responsive element‐ Iron‐responsive element‐ Blood 248 15q25.1 IREB2 registers cytosolic iron status Ziółkowska‐
binding protein 2 binding protein 2 mainly through an iron‐sulfur switch Suchanek
mechanism et al. (2015)
4. HHIP Hedgehog‐interacting Hedgehog‐interacting Plasma, 20 Mice 4q31.21 Hhiphaploinsufficiency leads to increased Wan et al. (2017)
protein protein urine, and lung susceptibility towards the development
tissue of emphysema following exposure to
chronic cigarette smoke (CS).
5. AGER Advanced glycosylation end Receptor for advanced NETT/NAS, – 6p21.32 RAGE has systemic anti‐inflammatory Kim and Do
product‐specific receptor glycan end GenKOLS, activity Lee (2015)
products (RAGE) ECLIPSE, and
COPD Gene
study cohort
6. CXCL8 C‐X‐C motif chemokine Interleukin‐8 – – 4q13.3 CXCL8 is a member of the chemokine Gilowska (2014)
ligand 8 family and is a major chemoattractant to
neutrophils
7. KCNE2 Potassium voltage‐gated Potassium Blood 9338 21q22.11 Modulates the gating kinetics and Cho et al. (2015);
channel subfamily E voltage‐gated channel enhances the stability of the channel Yang
regulatory subunit 2 subfamily E member 2 complex et al. (2004)
8. HTR4 5‐Hydroxytryptamine 5‐Hydroxytryptamine Lung tissue; fetal 3;12 5q32 This receptor is mediated by G proteins Hodge
receptor 4 receptor 4 tissue that stimulate adenylate cyclase et al. (2013)
9. GSTCD Glutathione Glutathione S‐transferase C‐ Human airway – 4q24 GSTCD lacks key functional domains Obeidat
S‐transferase C‐terminal terminal domain‐containing smooth muscle important for GST (Glutathione S‐ et al. (2013)
domain‐containing protein cells transferase) activity
10. TNS1 Tensin 1 Tensin‐1 Human airway – 2q35 TNS1 gene is associated with airflow Stylianou, Clark,
smooth muscle obstruction in GWAS and
cells Bradding,
(2016)
(Continues)
|
7
8 | WADHWA ET AL.

can be prevented by utilizing gene editing tools by modifying the genes

Cho et al. (2014)

which are involved in the diseased state.

et al., (2011)
et al. (2011)
Reference

Hersh

Kong
3.2 | Epigenetic modifications in COPD
Epigenetic modifications refer to alteration in the gene expression

the effect of BICD1 in emphysema could

interleukin‐2 dependent T‐cell growth.

pattern which may be due to histone modification, DNA methylation,

SOX5 SNPs have been associated with
pharmacogenetic potentially relevant

be related to shortened telomeres

TGF‐β 2 has suppressive effects on

gene silencing by Noncoding RNA, an aberration in chromatin and

DNA occurring due to environmental influences (Handy, Castro, &
Loscalzo, 2011). Evidence suggested that epigenetic changes asso-
Mechanism genes involved

ciated with COPD include DNA methylation at specific CpG Islands

as mentioned in Table 3, these studies are based on analysis of blood
sample for genotyping (Aggarwal, Wadhwa, Thapliyal et al., 2018). A
major cause of epigenetic changes in bronchial epithelium genes is
for COPD

suggested to be due to environmental stress or cigarette smoke

(Schamberger, Mise, Meiners, & Eickelberg, 2014) and the epigenetic
pathways regulate inflammation in the airways. Features of COPD
can be highlighted by epigenetic variation study, which could not be
Chromosomal

fully inferred by DNA sequencing technology, GWAS or meta‐

12p11.21

analysis. This study could help to identify susceptibility developed in

12p12.1
location
1q41

smokers, identification of post smoking cessation and continued risk

to develop COPD.
population
6663; 15
Study

3.2.1 | Epigenetic mechanism of COPD

2380
386

There are three levels of epigenetic gene regulation: DNA modifica-

Blood; Lung tissue
Sample analyzed

tion, histone modification and transcriptionally packed chromatin or

noncoding RNAs. These modifications are encountered and studied in
COPD models or patients (Schamberger et al., 2014).
Blood

Blood

DNA methylation in COPD

Transforming growth factor

Transcription factor SOX‐5

Typically, DNA methylation act by either gene activation or

repression. GWAS of large scale DNA methylation study indicates
Protein bicaudal D

349CpGs, to be significantly associated with COPD (Qiu et al., 2012).

It also suggests that several genes involved in COPD, are also in the
Protein name

homolog 1

inflammation pathway, stress response and healing wound. Gene

SERPIN 1 studied in a population of 1481 subjects where the
β‐2

genomic analysis DNA was extracted from white blood cells is found
in hypomethylation state in COPD patients, indicating gene activa-
Transforming growth factor

tion or over expression (Qiu et al., 2012). Several other studies

BICD cargo adapter 1

established DNA methylation in COPD for a variety of genes. One

such example is for hypomethylation of gene encoding‐1‐antitrypsin,
which is in significant correlated in COPD (Qiu et al., 2012; Siedlinski
Gene name

SRY‐box 5

et al., 2012). Gene expression level alters when DNA methylation

occurs in smoking related blood peripheral leukocytes and small cell
β‐2

airways, this leads to changes in GPCR15 (G Protein Coupled

(Continued)

Receptor) and NRF 2 (nuclear factor erythroid 2‐related Factor 2;

Vucic et al., 2014; Wan et al., 2012). Smoke related animal model
symbol
TGFB2

BICD1
SOX5
Gene

study on mice lung tissue sample and human BEAS‐2B lung epithelial
TABLE 2

cells reveals that the expression of HDAC6 increased hypomethyla-

tion in COPD, which can be linked to several other diseases as well
S.No
11.

12.

13.

(Lam et al., 2013).

T A B L E 3 Pathogenesis of COPD due to epigenetic modifications

Modifica-
WADHWA

tion Gene Population Chromosomal Mechanism genes

S.No Gene reported expression Protein Approach with COPD SNPs location involved Reference
ET AL.

1. SERPINA Hypome- Under α 1‐ antitrypsin Pi system 2; 1086 rs8004738 14q32.13 Protease inhibition DeMeo and Silverman,
1 thylation Expressed (electrophoresis), rs28929474 (2004); Yuan, Chang
GWAS and Deng (2009);
Eriksson, (2018); Li
et al. (2017)
2. SERPIN 2 N.A. Under Protease inhibitor Illumina array‐based 973 rs6734100 2q36.1 Protease inhibition Zhu et al. (2007)
Expressed 7; PI7 custom SNP rs729631
genotyping platform rs975278
rs7583463
rs6748795
3. CHRNA7 N.A. Over Neuronal GWAS 2614 4‐copy of CNV‐3956 15q13.3 Ligand‐gated ion Wang, Shi, Zhu, Gao,
Expressed acetylcholine increased COPD risk channel and Zhang, (2017)
receptor subunit
α‐7
4. CHRNA5 Hyperme- N.A. Neuronal GWAS and meta‐ 2614 rs17486278 15q25.1 Causes opening of Morrow et al. (2016);
thylation acetylcholine analysis. an ion‐conducting Nedeljkovic
receptor subunit channel across the et al. (2018)
α‐5 plasma membrane.
5. CHRNA3 Hyperme- N.A. Neuronal GWAS and meta‐ 2614 rs12914385 15q25.1 Causes opening of Cho et al. (2014); Lee
thylation acetylcholine analysis. an ion‐conducting et al. (2015);
receptor subunit channel across the Nedeljkovic
α‐3 plasma membrane. et al. (2018)
6. EGLN2 N.A N.A Egl nine GWAS 724 subjects, rs7937 19q13.2 Regulates Nedeljkovic
homolog 2 both blood and posttranscriptional et al. (2017)
lung tissue modifications of
sample hypoxia induced
factor
7. MMP1 N.A N.A Collagenase I Expression analysis in 63 N.A. 11q22.2 Cigarette smoke Gosselink et al. (2010);
transgenic mice responsive (CSR) Houghton (2015);
element within the Woode, Shiomi, and
promoter region of D'Armiento, (2015)
mmp‐1
8. MMP12 N.A N.A Fragments of Gene‐association 8300 rs626750 11q22.2 Elastase activity Houghton, (2015);
elastin study Hunninghake
et al., (2009)
(Continues)
|
9
 10

TABLE 3 (Continued)
|

Modifica-
tion Gene Population Chromosomal Mechanism genes
S.No Gene reported expression Protein Approach with COPD SNPs location involved Reference
9. VHL Hypome- Over von Hippel‐ Gene polymorphism 164;15 rs11549467 3p25.3 Involved in the Vucic et al. (2014); Yu,
thylation expressed Lindau disease study rs11549465 ubiquitination and Wang, and
tumor subsequent Cheng (2017)
suppressor proteasomal
degradation via the
von Hippel‐Lindau
ubiquitination
complex
10. MUC13 Hypome- Over Mucin−13 Illumina 15 N.A. 3q21.2 Epithelial and Vucic et al. (2014)
thylation expressed Infinium 27 K hemopoietic
Methylation Arrays, transmembrane
San Diego, CA mucin that may play
a role in cell
signaling.
11 NFE2L2 Hyperme- Under Nuclear factor genotyped at K‐ 15;1390 rs1806649 2q31.2 Transcription Figarska, Vonk, and
thylation expressed erythroid 2‐ Bioscience (UK) activator that binds Boezen, (2014); Vucic
related Factor 2 to antioxidant et al. (2014)
response (ARE)
elements in the
promoter regions of
target genes
12 MMP14 Hyperme- Under matrix TaqMan Genotyping 15;284 rs1003349rs1042703- 14q11.2 Endopeptidase that Hadchouel et al.
thylation expressed metallopepti- rs2236302r- degrades various (2008); Vucic
dase 14 s1042704rs2236303- components of the et al. (2014)
rs2236307 extracellular matrix
such as collagen.
Activates
progelatinase A.
13 CD9 Hyperme- Under CD9 antigen Illumina 15 N.A. 12p13.31 Involved in platelet Vucic et al. (2014)
thylation expressed Infinium 27 K activation and
Methylation Arrays, aggregation.
San Diego, CA Regulates paranodal
junction formation.
Involved in cell
adhesion, cell
motility and tumor
metastasis.
Required for sperm‐
egg fusion.
(Continues)
WADHWA
ET AL.
TABLE 3 (Continued)

Modifica-
WADHWA

tion Gene Population Chromosomal Mechanism genes

ET AL.

S.No Gene reported expression Protein Approach with COPD SNPs location involved Reference
14. LGALS3 Hyperme- Under Galectin‐3 Illumina 15 N.A. 14q22.3 Galactose‐specific Vucic et al. (2014)
thylation expressed Infinium 27 K lectin which
Methylation Arrays, binds IgE.
San Diego, CA
15. TFF3 Hyperme- Under Trefoil Factor 3 Illumina 15 N.A. 21q22.3 Involved in the Vucic et al. (2014)
thylation expressed Infinium 27 K maintenance and
Methylation Arrays, repair of the
San Diego, CA intestinal mucosa.
Promotes the
mobility of epithelial
cells in healing
processes (motogen)
16. PTEN Hyperme- Under Phosphatidylino- Oligo Pool (OPA) by 15;53 rs701848rs1903858 10q23.31 Tumor suppressor. Hosgood III, Menashe,
thylation expressed sitol 3,4,5‐ the Illumina Acts as a dual‐ He, Chanock, and Lan,
trisphosphate 3‐ GoldenGate Assay specificity protein (2009); Shi et al.
phosphatase and phosphatase, (2012); Vucic
dual‐specificity dephosphorylating et al. (2014)
protein tyrosine‐, serine‐ ,
phosphatase and threonine‐
PTEN phosphorylated
proteins
17. MUC1 Hyperme- Under Mucin‐1 Genome‐wide 15 rs4971100 1q22 The α subunit has cell Vucic et al. (2014)
thylation expressed association adhesive
studies (GWAS) properties.The β
subunit contains a
C‐terminal domain
which is involved in
cell signaling,
through
phosphorylations
and protein‐protein
interactions.
18. CKB Hyperme- Under Creatine Kinase B Illumina 15 N.A. 14q32.33 Reversibly catalyzes Vucic et al. (2014)
thylation expressed Infinium 27 K the transfer of
Methylation Arrays, phosphate between
San Diego, CA ATP and various
phosphogens
(Continues)
|
11
 12

TABLE 3 (Continued)
|

Modifica-
tion Gene Population Chromosomal Mechanism genes
S.No Gene reported expression Protein Approach with COPD SNPs location involved Reference
19. BNIP3 Hyperme- Under BCL2/adenovirus Illumina 15 N.A. 10q26.3 Apoptosis‐inducing Vucic et al. (2014)
thylation expressed E1B 19 kDa Infinium 27 K protein that can
protein‐ Methylation Arrays, overcome BCL2
interacting San Diego, CA suppression
protein 3
20. TTC3 Hyperme- Under E3 ubiquitin‐ Illumina 15 N.A. 21q22.13 Mediates the Vucic et al. (2014)
thylation expressed protein Infinium 27 K ubiquitination and
ligase TTC3 Methylation Arrays, subsequent
San Diego, CA degradation of
phosphorylated Akt
(AKT1, AKT2 and
AKT3) in the
nucleus
21. SMPD3 Hyperme- Under Sphingomyelin Illumina 15 N.A. 16q22.1 Catalyzes the Vucic et al. (2014)
thylation expressed phosphodiester- Infinium 27 K hydrolysis of
ase 3 Methylation Arrays, sphingomyelin to
San Diego, CA form ceramide and
phosphocholine.
22. CXCL1 Hyperme- Under Growth‐ Illumina 15 N.A. 4q13.3 Chemotactic activity Vucic et al. (2014)
thylation expressed regulated α Infinium 27 K for neutrophils
protein Methylation Arrays,
San Diego, CA
23. EPHX1 Hyperme- Under Epoxide Geneservices Ltd 15; 1017 rs2854450 rs2854451 1q42.12 Biotransformation Chappell et al. (2008);
thylation expressed hydrolase 1 (Cambridge, UK) rs3753658rs1051740 enzyme that Vucic et al. (2014)
using Taqman rs2234922 catalyzes the
assays hydrolysis of arene
and aliphatic
epoxides
WADHWA
ET AL.
WADHWA ET AL.
Histone modification
Inside the cell nucleus, the DNA strand is folded and wrapped around an
octamer complex of a protein consisting histone protein, these combine
and form a nucleosome. Amongst several histone modifications, the most
important is in the amino terminal of H3 and H4 through changes in
lysine residue by acetylation and methylation (Grewal & Moazed, 2003).
Mostly, increased acetylation corresponds with transcriptional activity,
while decreased acetylation connects with transcriptional inactivation
(Grewal & Moazed, 2003). Moreover, heterochromatin assembly is
strongly associated with methylation of histone H3 lysine 9 (Grewal &
Moazed, 2003). However, few post translational modifications are in
connection with gene expression either can be activated or repressed.
Histone acetylation/deacetylation
Gene transcription can be initiated by allowing chromatin remodeling
which regulates gene expression by acetylation of histone in the nucleus
to unfold the chromatin structure so that transcription factors and RNA
polymerase can bind to available unfolded DNA (Kouzarides, 2007;
Shahbazian & Grunstein, 2007). Multiple inflammatory mediators,
cytokines, and chemokines are involved in COPD inflammatory response,
which is generated by inflammatory cascade due to gene response
(Aggarwal, Wadhwa, Thapliyal et al., 2018). Histone acetylation control
these proinflammatory genes through regulating promoters of activator
protein (AP)‐1 and transcription factors NF‐kB. These pro‐inflammatory
cytokines play an important role by activating NF‐kB, activated NF‐kB
has the potential to bind to DNA at specific recognition sequence
enabling interaction with large coactivator molecules (Lawrence, 2009).
Coactivator molecule for instance, CRE binding protein, the p300
controls intrinsic activator of HAT. Therefore, the outcome of NF‐kB
activation is the increased rate of histone acetylation which leads to
altered inflammatory gene expression, similar is the case with gene
CXCL8 (P. Barnes, Adcock, & Ito, 2005). Several studies that reported,
NF‐kB is upregulated in smokers with COPD in different tissue samples
and bio‐fluids such as in lung homogenate (Szulakowski et al., 2006), rat
lungs exposed to smoke (Marwick et al., 2004), blood sample
(Zaynagetdinov et al., 2016), BALF fluid (Zaynagetdinov et al., 2016),
and so forth. Moreover, NF‐kB can be activated by oxidative stress and
tumor necrosis factor‐α (TNF‐α), over expressing the gene CXCL8 which
in turn increases the release of IL‐8 (Kode, Yang, & Rahman, 2006;
Rahman, Gilmour, Jimenez, & MacNee, 2002). Cigarette smoke activates
Mitogen and stress activated kinase 1 (MSK1), this MSK1 gets in contact
with a family member of NF‐kB RelA/p65, leading to acetylation and
phosphorylation which is studied on human bronchial epithelial cells. This
causes post translational modification of histones, phosphoacetylation of
histone H3, and acetylation of histone H4 (Sundar et al., 2012).
HDACs regulates proinflammatory cytokines negatively (Adcock,
Tsaprouni, Bhavsar, & Ito, 2007). Histone deacetylation may occur by
blocking the transcription of inflammatory genes. This blocking is
achieved as a result of interaction between NF‐kB family member
RelA/p65 with HDAC1 and HDAC2 (Ashburner, Westerheide, & Baldwin,
2001). In both cigarette smoker and excigarette smoker patients with
COPD, the activity of HDAC is reduced in samples of alveoli containing
macrophages, peripheral blood monocytes, and airway biopsies (Chen
| 13
et al., 2012; Ito et al., 2005; Szulakowski et al., 2006). Reduced HDAC is
related to the severity of COPD, with a low reduction in HDAC3 and
HDAC5 (Ito et al., 2005). In small airways, the reduced levels of HDAC2
were found to be more significant in cigarette smoking COPD patients
(Isajevs, Taivans, Svirina, Strazda, & Kopeika, 2011). Accordingly, CXCL8
histone acetylation increases in the airway biopsies sample of COPD
patients as a promoting factor for proinflammatory cascade initiation
(Isajevs et al., 2011; Ito et al., 2005). One of the reason for emphysema
can be HDAC inhibition, due to the fact that HDAC mechanism play a
pivotal role in adult lungs (Mizuno et al., 2010). When CXCL8 expression
is increased, HDAC activity decreases due to oxidative stress with the
help of HDAC2 tyrosine nitration and serine phosphorylation(Kode et al.,
2006; Marwick et al., 2004; Marwick et al., 2009) or by S‐nitrosylation of
HDAC2 cysteine (Malhotra et al., 2011). One of the inhibitors of HDAC,
MS‐275 when exposed to smoke and associate with anti‐inflammatory
cytokine IL‐10 (Leus et al., 2017). In another study, Clarke et al (2008)
reported that protein kinase C (PKC) β increases TNF‐α‐induced NF‐κB
transcriptional activity at the promoter of CCL11 through recruiting
p300/CBP‐associated factor and acetylation of histone H4 in human
airway smooth muscle cells. Hence, a response to cigarette smoke
corresponds to the upregulate acetylation of histone (H4) and in
exsmokers upregulated histone H3. Therefore, a misbalance created
between acetylation and deacetylation increases the probability of COPD
development (P. J. Barnes, 2009).
Sirtuins comprise of seven family of proteins SIRT1‐SIRT7, SIRT
belonging to NAD + family which are dependent deacetylase, SIRT is
known to regulate the mitochondrial functions and proteins while
existing in multiple subcellular locations (Finkel, Deng, & Mostoslavsky,
2009; Houtkooper, Pirinen, & Auwerx, 2012). Much is not known about
SIRT, but few proteins are studied for interaction, including transcription
factor, cellular proteins, signaling molecule and histone (Finkel et al.,
2009; Houtkooper et al., 2012). Cigarette smoke causes stress because of
the presence of oxidants or aldehydes, this stress causes covalent
modification of SIRT1 which reduces the enzymatic activity and initiates
proteasomal degradation (Arunachalam, Yao, Sundar, Caito, & Rahman,
2010; Caito et al., 2010). SIRT1 is also known to interact with NF‐kB
subunit RelA/p65, diminished levels of SIRT1 in the macrophages and
lungs of cigarette smokers are in correlation to COPD which represents
an increased rate of acetylation and further, increased IL8 release
(Rajendrasozhan, Yang, Kinnula, & Rahman, 2008; Yeung et al., 2004).
Decreased expression of SIRT1 is observed in the large airways of
smokers and in both large and small airways of COPD patients (Isajevs,
Strazda, Kopeika, & Taivans, 2012). Hence, SIRT1 plays a crucial role in
the regulation of NF‐kB dependent inflammatory cascade.
Histone methylation
Apart from histone acetylation and deacetylation, histone methyla-
tion is a complex mechanism where histone methyltransferase (HMT)
and histone demethylase (HDM) play a pivotal role in the regulation
of histone methylation (D. D. Wu et al., 2017). The complexity of
histone methylation can be studied as different and opposite
functional outcomes of histone methylation of residues such as
lysine or arginine can be obtained, additionally, the intensity of
14 | WADHWA
methylation may vary distinctly for the same residues (Mosamma-
parast & Shi, 2010). For for example, Histone H3 lysine 4
trimethylation (H3K4me3) is usually present in active genes while
methylations of H3K9 and H3K27 are in close proximity to gene
repression. Till now, limited research is focused on histone methyla-
tion mechanism in COPD. In COPD studies on histones, smoke
exposed mouse lungs have been collected and studied using the
spectroscopic method to recognize novel histone markers, it includes
acetylation, monomethylation, and dimethylation of histone H3 and
H4 significant residues lysine and arginine (Sundar, Nevid, Friedman,
& Rahman, 2013). Andresen, Günther, Bullwinkel, Lange, and Heine
(2011) reported the pathophysiological progression of the disease is
in tight coordination with elevated β‐Defensin 1 (DEFB1) mRNA
levels are found in association to H3K4me3 significantly identified in
bronchial epithelium and BALF obtained from 34 COPD subjects. In
addition, Yildirim et al (2006) found protein arginine methyltransfer-
ase 2 (PRMT2) protein level and mRNA are upregulated in the lung
tissue of hypoxic mouse lung tissue that is developing COPD.
Similarly Hussain et al (2009) reported histone H3K27me1 and
histone H3K27me2 can be recognized in the group exposed to
cigarette smoke. Hence, further studies and research is required to
identify the mechanisms involved in the pathogenesis of COPD.
Noncoding RNA
Several recent reports provided experimental evidence suggesting
the involvement of miRNA in inflammatory response via innate and
acquired immune response, additionally miRNA is also known to have
a pivotal role in sustaining homeostasis during lung development,
suggesting indirect association of miRNA in inflammatory lung
diseases, including COPD (Ezzie et al., 2011; Oglesby, McElvaney,
& Greene, 2010; O'neill, Sheedy, & McCoy, 2011). Recently several
authors reported that miRNA generally downregulates as an effect of
cigarette smoke (De Flora et al., 2012; Izzotti, Calin, Steele, Croce, &
De Flora, 2009; Pottelberge et al., 2011). Several studies identify
differentially expressed genes in the lung tissue of COPD patients in
comparison with healthy individuals (De Flora et al., 2012; Ezzie
et al., 2011; A. C. Hsu et al., 2017). A. C. Hsu et al (2017) identified
miRNA 125a and 125b involved in inflammation in vivo and ex vivo
studies on 15 COPD subjects, similarly, De Flora et al (2012)
identified 28 miRNAs in smokers, these miRNAs could be related to
the pathophysiology of COPD (Ezzie et al., 2011). A general
downregulation of miRNA expression in alveolar macrophages
derived from smokers compared to non‐smokers. Significantly lower
expression of miR‐452 has been linked to increased expression of
matrix metalloproteinase 12 (MMP12), analyzed from BALF of 24
COPD individuals which is known to be an important effector of
smoking‐related diseases (Graff et al., 2012; Hunninghake et al.,
2009). Recently Christenson et al. (2013) reported miRNA profiles
are altered with regional emphysema severity and can modulate
disease‐associated gene expression networks. The authors found that
the pathway gene sets involved in cell surface signaling and ECM
maintenance are inversely correlated with the expression of multiple
upregulated miRNAs. Particularly, this study identified a subset of
ET AL.
miRNAs, including miR‐30c, miR‐181d, and miR‐638, whose expres-
sion levels were associated with those of their mRNA targets
(Schamberger et al., 2014). They also propose that miR‐638 may play
an important role in COPD pathology by responding to oxidative
stress, and contribute to accelerated lung aging.
3.2.2 | Smoking independent epigenetic variants
of COPD
As COPD is a complex disease, several genes are differentially expressed
due to epigenetic modification irrespective of no smoking cases. These
genes are smoking independent epigenetic variants of COPD. For
example, Nedeljkovic et al (2017) genotyped 724 individuals on whole
blood genomic DNA for association with DNA methylation data in the
region 19q13.2 and conducted epigenome wide association analysis. They
found that gene Egl‐nine homolog 2 (EGLN2) and RAB4B (member RAS
oncogene family), are associated with COPD and not associated with
smoking, consistently associated with differential DNA methylation in
blood at four CpG sites. They additionally found methylation is also
associated with gene expression level in blood and in lung tissues
(Nedeljkovic et al., 2017). This suggests the importance of epigenetic wide
analysis to recognize the epigenetic changes in various genes, which can
be used for diagnosis or as a therapeutic target.
4 | G E N E E D I T I N G TE C H N O L O G Y
The phenomenon by which DNA can be engineered (insertion, deletion or
replacement) to understand the function of the gene. Tools for genome
editing includes conventional homologous recombination (HR) and
advanced tools including zinc finger nucleases (ZFNs), transcription
activator like effector nucleases (TALENs) and clustered regularly
interspaced short palindromic repeats (CRISPR)—Cas system which
potentially can be applied to human pluripotent stem cells (hPSCs)
wherein the template DNA can be exposed to natural mutations or the
mutations present can be repaired for creating isogenic control (Gaj,
Gersbach & Barbas, 2013). ZFNs and TALENs are simple to engineer and
have low cytotoxicity (Mussolino et al., 2011). CRISRP/Cas‐9 is favorable
because it is based on RNA to guide DNA in comparison to ZFNs and
TALENs which rely on proteins for specific target DNA. CRISPR/Cas‐9 is
more efficient, cost effective, and easy to use technology (Ding et al.,
2013). We shall briefly discuss the gene editing approaches as:
4.1 | Homologous recombination
The method of introducing a specific gene sequence into the host
genome is based on homologous recombination or gene targeting. In
a cell, when a double stranded DNA breaks, it can be repaired by
homologous or nonhomologous end joining (San Filippo, Sung, &
Klein, 2008). Homologous recombination results in crossing over
leading to genetic recombination or can occur by the introduction of
a transgene that integrates into the genomic DNA without the
assistance of nucleases.
WADHWA ET AL. | 15

4.2 | Zinc figure nucleases (ZFNs)
ZFNs consist of DNA binding domain and a DNA cleaving domain. The
binding domain is composed of three–six Cys2‐His2 repeats called zinc
finger, which recognizes cumulatively recognizes 9–18 bps of the DNA
(Porteus & Carroll, 2005). This leads to activation of dimerized
restriction enzyme II, Fok I which cleaves DNA with sticky ends.
Further, ZFNs stimulates DNA repair mechanism endogenously in the
presence of template donor DNA, which can lead to substitution of
target allele (Urnov, Rebar, Holmes, Zhang, & Gregory, 2010).
4.3 | Transcription activator –like effector
nucleases (TALENs)
TALENs are also composed of DNA binding domain called the TAL
repeats and a cleavage domain of Fok I dimer which cleaves the
double stranded DNA (Boch et al., 2009). TAL repeats contains 33–
35 highly conserved amino acids that target DNA. 12th and 13th
amino acids differ and are termed as repeat variable residues (RVD)
so that one repeat identifies one nucleotide (Boch et al., 2009).
Variation in RVD of the TAL repeats recognizes DNA sequence
specifically. On fusion with DNA generates TALE nucleases that
introduces foreign DNA for gene repair or disrupt endogenous gene
function (Cermak et al., 2011).
4.4 | Clustered regularly interspaced short
palindromic repeats (CRISPR)‐Cas system
Clustered regularly interspaced short palindromic repeats is based
on a repetitive pattern (24–48 base pairs) wherein the DNA
sequence would repeat itself in the reverse orientation after a
spacer DNA. The unique system was observed in bacteria and archea
as a means of adaptive immunity (Barrangou et al., 2007). Once the
bacterium is attacked by phage, initially the bacterium transcribes
the spacer and palindromic DNA into RNA. RNA III along with Cas 9
cleaves the RNA. The CRISPR RNA forms a dimer with transactivat-
ing crRNA, thus forming a guide RNA (gRNA; Jinek et al., 2012; Mali,
Yang et al., 2013). The gRNA directs Cas‐9 for endonuclease activity
by recognizing 20 bp target which further breaks the double
stranded DNA. The Cas‐9 comprises of two domains, a HNH domain
with breaks the complementary strand of DNA and RuvC‐like domain
that breaks the noncomplementary DNA strand (Jinek et al., 2012;
Mali, Yang et al., 2013). Thus, by using this technology gRNA can be
engineered complimentary to DNA sequence allowing simple and
rapid correction of defective genes to restore normal functioning.
5 | A P P L IC A T I O N S O F G E N E E D I T I N G
TECH NOL O GY IN COP D
Till now very limited research studies are available on the application
of gene editing technology in COPD (Figure 3).
A major risk factor for COPD is due to genetic variation in genes
aggregated in families. Growth differentiation factor 15 (GDF15),
which is a member of TGF‐β family, increases in COPD cigarette
smoker group in airway epithelium and human primary airway
epithelial cells (HAEC; Q. Wu, Jiang, & Chu, 2012). Human trachea‐
bronchial epithelial cells packaged with CRISPR/Cas9 targeting exon
1infected by lentivirus, for human the GDF15 gene, results in small
mutations such as deletion or insertion in exon 1 of the GDF15 gene
and this significantly reduces GDF15 mRNA, protein and cellular

F I G U R E 3 Strategies of the implication of genome editing technology in chronic obstructive pulmonary disease. Lung tissues,
bronchoalveolar lavage, and bronchial brushings obtained from patients with COPD are extrapolated for the identification of novel genes by
using microarray and proteomics technology. The identified novel gene can be rectified further by recently developed genome editing
technologies including Zinc finger nucleases (ZFN), transcription activator‐like effector nucleases (TALEN) and clustered regulatory interspaced
short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) systems for clinical applications for treatment of COPD. COPD: chronic
obstructive pulmonary disease [Color figure can be viewed at wileyonlinelibrary.com]
16 | WADHWA
senescence (Q. Wu et al., 2012). The first application of CRISPR/
Cas9‐mediated gene editing in HAECs had depicted the possibility of
increased research on COPD with gene editing. Another major
symptom of COPD pathogenesis is airway mucus hypersecretion
regulated by Mucus secretion regulator SAM‐pointed domain‐
containing Ets‐like factor (SPDEF) as key regulator of mucus
secretion by silencing its expression. Song et al (2017) reported that
gene SPDEF silencing can be reduced by using ZFPs and CRISPR/
dCas platforms, hence decrease in the mucus production in lung
epithelial cells. Therefore, we can say that epigenetic downregulation
of SPDEF possesses a vital role in the generation of a constant effort
of reduction in mucus, this may act as an upcoming therapeutic
approach for patients with excessive mucus secretion. Further DNA
targeting systems had been applied for genome editing approach, for
for example, V64 fused with zinc finger primers as an activator of
artificial transcription, which binds to gene promoter of γ‐globin, for
developmental silencing of fetal γ‐globin in primary human adult
erythroblasts (Wilber et al., 2010). Furthermore, TALENs are
designed such that TALEs fuses with DNMT3A or TET1 and targets
Ascl1 in neural stem cells in the promoter region, affecting the
variation in gene expression, and methylation (Lo, Choudhury,
Irudayaraj, & Zhou, 2017).
6 | LIMITATIONS AND FUTURE
PE RSP ECTIVE
In recent years, ZFNs, TALENs, and CRISPR/Cas9 are the gene
editing technologies discovered with increased efficiency. Due to
active challenges related to engineering associated with ZFNs and
TALENs, CRISPR/Cas9 is considered to be an efficient gene editing
tools since its discovery in 2013. The simplicity and rapid application
of CRISPR/CAS9 highlighted and overshadowed ZFNs and TALENs.
Despite possessing wide application of gene editing technology there
are several limitations associated with ZFNs, TALENs, and CRISPR/
Cas9 such as off‐target effects, due to off target unwanted gene
mutation, or rearrangement may occur, which can cause risk of
oncogenesis, dysregulation in cell functioning, affecting the prolifera-
tion ability, and may lead to cell death. Hence, researchers are
working to improve technology by avoiding the problem of off‐target
effect, it is overcome by considering mismatch tolerance and NGG
mutations (P. D. Hsu et al., 2013; Pattanayak et al., 2013) and with
the introduction of offset nicking through Cas9 nickases and paired
gRNA (Mali, Aach et al., 2013; Ran et al., 2013), dose standardization
of Cas9, and gRNA are also to be accomplished (P. D. Hsu et al.,
2013) by swapping protospacer adjacent motif (PAM) specificity to
noncanonical sequences (Kleinstiver et al., 2015; Zhang et al., 2014)
and merging catalytically inactive Cas9 to Fok I (fCas9; Guilinger,
Thompson, & Liu, 2014). These methods can act in prevention of off‐
target risks but cannot completely eradicate them. Therefore it is
crucial to identify off‐site targets, which can be detected using high
throughput technologies such as genome wide association studies,
next generation sequencing, and so forth. Other challenges in
ET AL.
utilizing gene editing technology as therapeutics are a delivery
strategy, clinical settings, dose optimization, immune response,
pharmacokinetic analysis, biochemical analysis, long term effect,
and so forth laboratory systems are designed such that the targeting
could be studied in the more efficient way. For for example, IREB2 a
significant iron binding protein producing gene is dysregulated in
COPD, using technology such as CRISPR/CAS9 knockouts for
identification of gene importance and metabolism. The genome
editing approach for COPD can be used by targeting the exon in
15q25 locus having CHRNA3/CHRNA5 located on the same locus as
of IREB2. CHRNA3/CHRNA5 are usually found to be epigenetically
modified in COPD (Hardin et al., 2012). These studies may lead to the
development of therapeutically applicable solutions. The authors
would like to mention that gene editing technology has a huge
potential in therapeutic applications of COPD and other respiratory
disease. Recognition, optimization, and delivery are the three key
components for the development of gene editing technology
for COPD.
7 | CO NCL USION
COPD with huge global burden becomes an important disease for
the development of novel therapeutics and diagnostics. In this
review, we discussed the crucial genes and airway stress
biomarkers in detail to identify the genes or targets, which can
be used for genome editing approach. Genome editing approach
includes upcoming pioneer technologies ZFNs, TALENs, and
CRISPR/Cas9 to develop the system in which the disease can be
cured by targeting DNA. There are several drawbacks in these
technologies which are being worked to improve and irradiate off‐
target risks. So we can conclude that, before moving towards RNA
editing, gene, and protein targets are important for the develop-
ment of therapeutics and diagnostics.
AC KNO WL EDG EM E NT
PMH is funded by a Fellowship from the National Health Medical
Research Council, NHMRC (1079187). The authors acknowledge
NHMRC and the Graduate School of Health (GSH), University of
Technology Sydney (UTS), NSW, Australia for the funding.
CON F LI CT OF IN TE RES T S
The authors declare that there are no conflict of interests.
AU TH OR CO NTRIB UTION S
R. W., K. D., P. M. H., and P. K. M. conceived the idea, and all the
authors contributed to the preparation and editing of this manuscript
for intellectual content. All the authors read and approved the final
version of the manuscript.
WADHWA ET AL.
OR CID
Ridhima Wadhwa http://orcid.org/0000-0001-8074-1543
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SU PP ORT IN G IN FOR M ATI O N
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
How to cite this article: Wadhwa R, Aggarwal T, Malyla V,
et al. Identification of biomarkers and genetic approaches
toward chronic obstructive pulmonary disease. J Cell Physiol.
2019;1–21. https://doi.org/10.1002/jcp.28482