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Trigger of Autoimmune Diseases (SLE): Identification of LINE TranspositionBased Novel Therapeutic Molecular Targets
Anupama Tiwari, Upendra Kumar Soni
PII: S0306-9877(14)00392-2DOI: http://dx.doi.org/10.1016/j.mehy.2014.10.019Reference: YMEHY 7739
To appear in: Medical Hypotheses
Received Date: 2 August 2014Accepted Date: 23 October 2014
Please cite this article as: A. Tiwari, U.K. Soni, Trigger of Autoimmune Diseases (SLE): Identification of LINETransposition Based Novel Therapeutic Molecular Targets, Medical Hypotheses (2014), doi: http://dx.doi.org/10.1016/j.mehy.2014.10.019
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Page 1 of 18
Trigger of Autoimmune Diseases (SLE): Identification of LINE Transposition
Based Novel Therapeutic Molecular Targets
Anupama Tiwari 1 (M.Sc.) and Upendra Kumar Soni
1 (M.Sc.)
1Department of Molecular and Human Genetics, Banaras Hindu University, Varanasi
INDIA 221005
Upendra Kumar Soni
Department of Molecular and Human Genetics
Banaras Hindu University
Varanasi, INDIA 221005
+91-8052697657
upendrasoni20@gmail.com
Page 2 of 18
Trigger of Autoimmune Diseases (SLE): Identification of LINE Transposition Based Novel
Therapeutic Molecular Targets
Abstract
Autoimmune diseases are the highly heterogeneous at cellular and molecular level. The causes
and consequences of most of the autoimmune diseases are now well explored. However the
researches focusing on the development of biomarkers for the diagnosis of autoimmune diseases
are seems to be inadequate and given treatment are insufficient to control or cure the disease
properly. It is a big obstacle to develop any therapy without knowing the actual cause and
molecular event playing role in disease onset. In this article we are raising the involvement of
LINE or other transposition as a first trigger and cause for autoimmune disease. Further we are
proposing a novel hybrid aptamers based biocapturing model which would help in the
investigation of genome-wide LINE transposition in pristane induced SLE mice model.
Importantly the effect of new LINE movements at the expression pattern of neighboring genes
would be used as novel molecular prognostic biomarkers for onset of SLE and related
autoimmune diseases. We also proposing that the differential expression either inductive or
suppressive pattern of expected several candidate genes would be implicated in the defective
biochemical or cellular defects, and targeted therapy would be employed to such life threatening
disease.
Introduction
Autoimmunity diseases are one of the most leading causes of clinical complications and death
worldwide. In India and other developing countries, the mortality rate is high due different
autoimmune diseases in comparison to various infections [1]. Some of common autoimmune
diseases are rheumatoid arthritis, multiple sclerosis, juvenile diabetes, cardiomyopathy,
antiphospholipid syndrome, Guillain-Barré syndrome, Crohn's disease, Graves' disease, Sjogren's
syndrome, alopecia, myasthenia gravis, lupus erythematosus, and psoriasis, lack proper
treatment. Autoimmune diseases are more prevalent in women than men [2-5]
Developments of accurate and effective diagnosis methods for risk and onset of autoimmune
diseases in susceptible individuals are principally require. In this article we are suggesting a
Page 3 of 18
model to control auto immune burden by taking SLE as example. Regarding SLE, nucleic acid
antigen based biomarkers are very useful in diagnosis, evaluation, and management of SLE and
helps in detection of a disease flare and monitoring disease activity. An ideal biomarker should
accurately detect disease activity and guide therapy at every stage of SLE. From several decades
SLE diagnosis is based on the presence of auto-antibodies like ANA (antinuclear auto-
antibodies), which further has been sub grouped into dsDNA and ENA. Table 1 is showing the
suggested treatment SLE diagnosis based on presence of different types auto-antibodies against
the nuclear antigens and their association with various organs and their symptoms [6]. However
ANA has a low specificity for SLE, and it has very low sensitivity may be as low as 70%,
especially early in the disease. Anti-dsDNA is better with 95% specificity but has a low
sensitivity. So these two biomarkers are not effective during early time period of SLE diagnosis
in suspected individuals [7-8]
Some other biomarkers for SLE are also reported which includes auto antibodies for RNPs and
nuclear materials. Anti-C-1q antibodies, NMDA receptor antibody, and anti-alpha actinin
antibody have also used in early diagnosis of SLE and diagnosis of flares [9-11]. The recent
advances in lupus research has enhanced our knowledge and understanding, however, the
basic mechanism employed for disease initiation and progression is largely unknown and not
well understood therefore there is no effective diagnosis and cure for lupus so far. The
major treatment options include immunosuppressive drugs and steroids (orticosteroids, anti-
malarials, aspirin, and hydroxychloroquine,) that may put the patients at the risk to develop more
infections and other side effects. The need of a specific and side effects free therapeutic
approach is well felt among the scientists and clinicians. The autoimmune patients exhibit
tremendous heterogeneity in terms of clinical manifestations and serological patterns. This
heterogeneity poses a substantial challenge in identifying therapy suitable to different
subsets of SLE patients. For proper diagnosis and treatment strategies there is a
compelling need for the identification of specific molecular biomarkers associated with
development of specific end organ disease in SLE.
In this article we aim to understand different genetic and epigenetic regulatory mechanisms
operative in autoimmune disease initiation. The LINE transposons are now well known to
identify the global gene signature variation and as an initial step in identifying molecular
Page 4 of 18
biomarkers associated with disease development, progression and outcome of the treatment [12].
Immune responses are controlled by gene expression via multiple layers, including
chromatin remodeling, transcription factors, alternative splicing an d miRNA mediated
regulation. Investigations have shown that environmentally driven DNA methylation may
contribute to the etiology of SLE and seems be a crucial factor in SLE disease initiation and
progression [13-16].
The LINE transposition mediated control occupies a special regulatory status on the intensity and
duration of immune responses and tends to change gene expression to a modest level. The fine
tuning of LINE transposition regulation results in a quantitative effect on immune responses and
controls the intensity. It is estimated that transposition may regulate 30–50% of protein-encoding
genes [17-22]. In addition, unfortunately no such studies on gene expression or their
regulation by LINE transposition with Indian patients who are genetically different from
western populations are available. As the genetic background and ethnicity are the critical
factors affecting the disease course, the studies on global gene expression and regulation
with Indian autoimmune disease patients are warranted.
LINE transposition would be a first event to start the activation of the complex cascade for the
SLE onset. In this context, here we are proposing a novel technical approach which will explore
the role of LINE transposition and SLE trigger. To achieve this goal, aptamers would be
modified and used as transposition capturing device during in-vivo SLE induction in mice
models.
Aptamers are single stranded DNA or RNA ligands which can be selected for different targets
starting from a huge library of molecules containing randomly created sequences. Aptamers are
being used as important component in medical researches and a preferable choice as biosensors
[23]. Some works aiming at developing aptamers-based biosensor for small molecules have been
reported which take advantage of the versatility and the flexibility of aptamers. Aptamers are
now able to recognize many of bio-molecules, and the hybrid technology has been using widely
to generate such hybrid Aptamers which possess the multiple specific recognition domains [24].
Here we are discussing such unique properties of aptamers for sensing, recognition and signal
relay for detection of LINE transposition in pristine induced SLE mice model.
Page 5 of 18
Hypothesis
We propose here aptamer based novel approach which permits to find out the LINE transposition
during triggering of SLE and or other autoimmune disease if transpositions are occurring. This
approach may offer a more targeted diagnosis and treatment minus the side effects associated
with conventional treatments for autoimmune diseases.
Evaluation of the hypothesis
In-Silico analysis of LINE distribution in genome of control group (Human and mouse)
Characterization of Long interspersed nuclear elements distribution in the genome of healthy
control groups (Human) or normal mouse model, is very critical step before observing the
LINE transposition in autoimmune diseases. LINE arrangement into genome will give us a
blue print for normal condition, which arrangement will use as reference-map for any
mobilization and insertion of LINE into new location of induced SLE disease mouse
model. Several bioinformatics tools are available which will be used for LINE distribution
analysis. Some of bioinformatics tools are listed in Table 2 [25]. By using suitable tool we shall
analyzed the LINE distribution in the genome and their chromosomal location, neighboring
genes in healthy control group (human) or normal mouse. Steinhoff C and Schulz W A has been
analyzed the human genome and found 177 putative autonomously active LINE-1 elements in a
healthy individual (Table-3). The complex arrangement of transposable elements in genome will
be annotated and used as for further experiments when aptamer based bio-capturing will be done.
The expected LINE movement to the new chromosomal location during triggering of
autoimmune diseases will be compared with this generated normal LINE distribution map.
Generation of specific aptamers with dual affinity for LINE element and transposes enzyme
Aptamers are random peptide, DNA or RNA fragments which have specific affinity for a target
molecule. Presently aptamers are being used in various aspects of medical researches [26]. They
are used as biomarkers, as carriers and for cellular localization as well. The aptamers
Page 6 of 18
synthesized by SELEX enrichment process. In this technique, the random nucleic acid fragments
are incubated with target molecules and the unbounded molecules are washed out, further the
bounded fragments are amplified which now become specific Aptamers for a specific
target molecule. Figure 1 is showing general schematic steps of aptamer synthesis by SELEX
protocol [27-32]. Importantly in our proposal LINE-transposition will be captured if
transposition involves in triggering of autoimmune diseases. For this purpose we require such
incredible aptamers which will bind with the LINE sequences when they are getting transposed
from one location to another location. During transposition the transposes enzyme makes RNA
intermediate and helps in the generation of new LINE DNA. New LINE DNA get integrate into
new location, this integration is also mediated by transposes enzyme. On this basis we have
decided that aptamers must be constructed with dual specificity which will recognize only the
transposes associated LINE sequences.
Dual specificity hybrid aptamers will comprise of transposes specific antibody and LINE
sequence specific nucleic acid moiety [24, 33]. This hybrid aptamer will possess two domains:
one is Transposes recognition domain and second is LINE sequence binding domain. Further this
hybrid aptamer will be tagged with confirmation dependent fluorescence moiety which will
illuminate when both domain will bind to their specific targets in cis format: transposes
enzyme and LINE sequence. This fluorescence tagged hybrid aptamer will make it easy
to capture the process of transposition outside and inside the cell as well . The proposed
model for generation of fluorescence tagged hybrid aptamer with dual specificity has been
presented in Figure-2.
Specific-confirmation activated fluorescence- hybrid aptamers with dual affinity for transposes
enzyme and LINE sequences will be used as a ‘biocapturing device’ which offer us to track the
LINE mediated transposition into genome at any time. As suspected LINE transposition may be
involved in the triggering of autoimmune disease but it is more important to know that where
transposition is happening in the genome?? By using such kind of approach these valuable
hybrid aptamers may serve as very important tools for looking after the genome architecture in
various autoimmune diseases.
Biocapturing of LINE transposition as trigger for autoimmune diseases by using
generated specific hybrid aptamers.
Page 7 of 18
After getting such incredible aptamers, triggering of autoimmune diseases by LINE
transposition in genome would be clearly visualized. This visualization will be done in an
inducible mouse model of SLE [34-37] and proposed steps will be involved for biocapturing of
LINE transposition in to pristane induced lupus model at different time points of induction. Same
strategy will also apply for human samples as well, at different period of onset and progressive
stages of any autoimmune disease.
Pristane induced mouse takes 10-14 days for showing the symptoms of the SLE, which indicate
us that for capturing the LINE-transposition we should be very precise with SLE induction time.
For this we have to capture transposition at the various time points for disease onset: 0 day to 14
day and for disease progression: 14 day to onward which will depend on the severity of the
autoimmune diseases. Figure 3 representing the step-wise procedure for LINE transposition
biocapturing.
Proposed mechanism
Each day (up to 14 days), the genomes of the pristane induced mice will fixed by chemical
treatment (formaldehyde or methanol) expecting intact DNA-protein complexes, followed by
slight membrane disruption. Now DNA-protein complexes will be incubated with fluorescence
tagged hybrid aptamers with dual specificity. Expected LINE DNA sequence- transposes
enzymes will be recognized by these aptamers and generated fluorescence will confirm its cis-
format (Figure 2). Genome fragmentation will be done for appropriate time period with optimum
sonication pulses.
Pulling down of entire aptamer-LINE DNA-transpose complexes: Aptamer specific affinity
columns will provide a way to separate and pull those fragments which are actually bounded
with LINE DNA-transposes complexes. Now the pulled fragments will be treated with
appropriate buffer which will allow dissociation of the aptamers followed by protease treatment
to remove transposes enzymes. Finally the captured DNA fragments will be sequenced by cyclic
array sequencing.
Cyclic array sequencing of captured DNA fragments
Page 8 of 18
After capturing the specific LINE sequences by using hybrid aptamers, we wish to know
the site and location where these LINE sequences are moving after triggering of the
pristane induced SLE and its progression. To achieve this goal we have to use cyclic array
sequencing approach in which the sequencing will be done by amplification of the DNA
fragments. This amplification makes the sequences more accurate. In cyclic array sequencing
the both end of fragmented DNA will be ligated with specific adaptors which will be done on a
micro chip. After adaptor ligation the primers which are specific for adaptor will start the
synthesis of the entire ligated DNA fragments. Importantly dNTPs will be differentially
fluorescence tagged. After incorporation of a single dNTP on the chip fluorescence will be
captured by UV illumination which will produce the color of respective tagged fluorescence
compound. The image will be analyzed by analyzer and each spot will write in the nucleotide
sequence format by computer programme [38-42]. After completion of the sequencing the
further bioinformatics analysis will be done to find out that what these sequences are?
BLAST will be performed with data bases which will indicate their chromosomal location in the
genome.
Annotation of captured sequences with database and characterization of their chromosomal
location (target genes)
During disease triggering, onset and their progression specific interplay of genes are required. To
know those genes and their expression pattern at various stages in autoimmune disease is very
critical. Further level of complexity generated when the post transcriptional gene regulation by
RNAi or miRNA occurs. One should be very careful when the complex disease is under
investigation for gene location, their expression pattern and gene regulation. Considering the
LINE transposition in SLE, it’s very important to know that at which chromosomal
location and beside which genes they are getting inserted. The LINE insertion into new site may
affect the expression of neighboring genes. The captured DNA fragments will allow us to find
out the LINE movement and their favorite chromosomal location. The LINE distribution of
healthy control group now will act as a reference for diseased group. LINE is moving from old
location (adjacent to gene 1 and gene 2) to new location of different chromosome where
adjacently two hypothetical genes (Gene 3 and Gene 4) are present (Figure 4). If somehow this
LINE insertion is going to affect the expression pattern of adjacent genes possibly this
Page 9 of 18
changed expression may contribute in the triggering, onset and progression of autoimmune
disease? After biocapturing of genome-wide LINE transposition during autoimmune disease
triggering and various stages of disease, several critical candidate genes will be highlighted.
Further the expression profiling of these important genes at either mRNA or protein expression
level will be done in an array fashion. Selected probes (RNA or antibody) for candidate
genes will be spotted on the microarray chip, which will be hybridized with the total
RNA or total protein from control and autoimmune disease groups. The chip will be
analyzed and their differential expression pattern will be resolved.
Expression pattern of target genes as biomarkers for autoimmune disease onset
During this study if any gene or set of genes and their expression pattern at mRNA or
protein level is found to be unique which makes them different from control group would be
a suitable biomarkers for respective autoimmune disease. This pattern may be commercialized as
detection and prognostic kit for various stages of the respective disease.
Implication of the hypothesis
As the etiology and causes of the autoimmune disease is not explored, this proposal may uncover
the role of LINE transposition in triggering of SLE and in same way other autoimmune disease.
The chromosomal location and expression of adjacent genes where new LINE sequences will
move would cross talk each other and may contribute in the disease onset also be discovered.
Considering the complexity of autoimmune diseases, here the hybrid aptamers would be a
novel and advanced molecular devices which are able to capture molecular event in whole
genome and their location as well.
The biocapturing of LINE transposition will result in the identification of new therapeutic targets
which are involve in the autoimmune disease. Further the change in the expression pattern of the
possible candidate genes would be the basis for prognostic molecular biomarkers which may use
in early diagnosis of the risk for the autoimmune diseases. Here the pristane induced SLE mouse
model will provide us an opportunity to look after the changes in the pattern of LINE movement
and the effect of transposition on the adjacent and trans-gene expression also in temporal
manner. Human SLE patients and different subsets will also undergo for investigation will
Page 10 of 18
provide us important information among them related to the disease triggering and progression
mediated by LINE movement.
Not only the SLE, other autoimmune disease can also be investigate by using same approach.
The other transposable elements are also can be targeted and according hybrid aptamers can also
be generated By such kind of study the novel molecular operations come in knowledge
which is still not explored in various lives threatening auto immune disease onset and
progression. The underlying mechanism and the signaling will be explored and offer novel
therapeutic targets for untreated autoimmune disease.
Conflict of interest statement
None
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Figure legends
Figure-1: Representation of SELEX enrichment process for aptamers synthesis.
Page 14 of 18
Figure-2: Generation of fluorescence tagged hybrid Aptamer with dual affinity for both
Transposes and LINE sequences and proposed mechanism for LINE transposition
biocapturing. Step 1-Showing the tagging of random DNA Aptamers with confirmation
activated fluorescence moiety along anti transposes antibody. Step 2- fluorescence tagged
hybrid Aptamers are incubated with the transposes bound LINE sequences. The Aptamers
which are heaving specific affinity for Transposes and LINE sequences in a Cis format only they
are showing fluorescence, rest of bounded Aptamers which don’t have confirmation activated
fluorescence properties they are getting washed out. Now finally specific – confirmation
activated fluorescence- hybrid Aptamers with dual affinity will be enriched by SELEX protocol.
Figure-3: Schematic representation of sequential events for LINE transposition biocapturing.
Pristane injection will given to the mice for SLE symptoms follow by peripheral blood or specific
cells collection from SLE mouse model or directly from human SLE patients will be done.
Fixation of the Genome and physically associated proteins/ enzymes will be done by chemical
treatment. After that the hybrid aptamers will be incubated with fixed genome-protein
complexes for desire time. The fragmentation of genome will be performed for the shortening
of the entire genome follow by pulling down the aptamers bounded LNE-transposes complexes
and removal of unbound molecules. Protease treatment will be done for the removal of the
transposase and the generated DNA fragments will be sequenced by the cyclic array sequencing
method.
Figure-4: Model for LINE movement and their impact on other genes and their expressions.
Page 15 of 18
Table 1: Clinical profile of SLE patients (Chauhan et al. 2013)
Age
(years)
Anti-
dsDNA
Anti-
ENA
Clinical Manifestations
Medications
35 + - Glomerulonephritis, Pericarditis,
Hepatomegaly
Carvidilol, Ramipril,
Lasilactone
50 + - Arthritis, Oral ulcer, Cuteneous Esomeprazole
28 + - Arthritis,Cutaneous NSAID
32
+
-
Pleuritics, Arthritis, Cutaneous,
Oral Ulcer
Prednosolone, HCQ
37 + - Arthritis, Cutaneous Prednisolone
28
+
-
Glomerulonephritis, Leucopenia,
Anemia
Prednisolone
22 + - Myositis Prednisolone, HCQ
45 + - Arthritis, Cutaneous NSAID
17 + - Arthritis, Oral ulcer, Cutaneous Prednisolone
40 + - Glomerulonephritis, Arthritis Prednisolone, HCQ
16
+
-
Arthritis, Cutaneous, Oral Ulcer,
Thrombocytopenia
Prednisolone
36 + - Oral Ulcer, Cuteneous, Arthritis Phentermine
25 + + Arthritis, Cuteneous, Oral Ulcer Prednisolone, HCQ
18 + + Glomerulonephritis, Cutaneous Prednisolone, HCQ
25 + + Glomerulonephrits, Arthritis, Oral
Ulcer
Prednisolone, NSAID
16
+
+
Cutaneous
Eltroxin, Mysolone
36 + + Arthritis Prednisolone, HCQ
28 + + Glomerulonephritis, Anemia Prednisolone
22
+
+
Glomerulonephritis, Arthritis,
Cutaneous
NSAID, HCQ
32 + + Arthritis, Cutaneous Prednisolone
32 + + Arthritis, Cutaneous Prednisolone
20 + + Arthritis, Cutaneous Prednisolone
27 + + Arthritis, Oral ulcer, Cutaneous Prednisolone
40 - + Glomerulonephritis, Cutaneous N.A.
24
-
+
Arthritis, Cutaneous, Oral ulcers
Alfacalcidol, Cosval PC
28
28
-
+
Oral ulcers, Cutaneous,
Leucopoenia
Prednisolone
36 - + Arthritis, Cutaneous, Oral ulcer, Prednisolone
Page 16 of 18
Anemia
42 - + Arthritis, Cutaneous, Oral ulcer Prednisolone, HCQ
32 - + Myositis, Arthritis Prednisolone, HCQ
36 - + Arthritis, Cutaneous Prednisolone
26 - + Pericarditis, Arthritis Prednisolone
32
-
+
Glomerulonephritis, Arthritis,
Cutaneous, Leucopenia
Prednisolone
18
-
+
Neurological symptoms,
Cutaneous, Hepatomegaly,
Splenomegaly
Prednisolone, Epsolin
34 - + Neurological symptoms, Arthritis, Cutaneous, Oral ulcers, Leucopenia
Prednisolone, NSAID
19 - + Arthritis Prednisolone, HCQ
Page 17 of 18
Table 2: Computational resources for TE (Transposable Element) discovery and TE
detection
Resource Location
BLASTER suite http://urgi.versailles.inra.fr/development/blaster/
Censor http://www.girinst.org/censor/download.php
find_ltr http://darwin.informatics.indiana.edu/cgi-bin/evolution/ltr.pl
FINDMITE http://jaketu.biochem.vt.edu/dl_software.htm
HMMER http://hmmer.janelia.org/
LTR_FINDER http://tlife.fudan.edu.cn/ltr_finder/
LTR_STRUC http://www.genetics.uga.edu/retrolab/data/LTR_Struc.html
LTR_MINER http://genomebiology.com/2004/5/10/R79/suppl/s7
LTR_par http://www.eecs.wsu.edu/ananth/software.htm
MAK http://wesslercluster.plantbio.uga.edu/mak06.html
MaskerAid http://blast.wustl.edu/maskeraid/
mer-engine http://mer-engine.cshl.edu/mer-home.php
mreps http://bioinfo.lifl.fr/mreps/
PILER http://www.drive5.com/piler/
PLOTREP http://repeats.abc.hu/cgi-bin/plotrep.pl
RepBase http://www.girinst.org/
RepeatFinder http://cbcb.umd.edu/software/RepeatFinder/
RepeatGluer http://nbcr.sdsc.edu/euler/intro_tmp.htm
RepeatMasker http://www.repeatmasker.org/
RepeatRunner http://www.yandell-lab.org/repeat_runner/index.html
RepeatScout http://repeatscout.bioprojects.org/
repeat-match http://mummer.sourceforge.net/
REPuter http://www.genomes.de/
RetroMap http://www.burchsite.com/bioi/RetroMapHome.html
SMaRTFinder http://bioinf.dimi.uniud.it/software/software/smartfinder
Tandem Repeats Finder http://tandem.bu.edu/trf/trf.html
Transposon Cluster
Finder http://www.mssm.edu/labs/warbup01/paper/files.html
TE nest http://www.plantgdb.org/prj/TE_nest/TE_nest.html
TRANSPO http://alggen.lsi.upc.es/recerca/search/transpo/transpo.html
TSDfinder http://www.ncbi.nlm.nih.gov/CBBresearch/Landsman/TSDfinder/
Tu LabTE tools http://jaketu.biochem.vt.edu/dl_software.htm
WU-BLAST http://blast.wustl.edu
Page 18 of 18
Table 3: Summary of 177 putative autonomously active LINE_1 elements (Steinhoff C and
Schulz W A 2010)
Chromosome #putative
Functional*
#Putative functional LINE-
1/108 bps*
1 12 4.86
2 10 4.15
3 11 5.64
4 17 8.85
5 15 8.29
6 14 8.22
7 4 2.54
8 13 9.04
9 5 3.78
10 3 2.23
11 11 8.00
12 9 6.85
13 5 4.41
14 5 4.79
15 6 6.05
16 5 6.12
17 6 7.50
18 4 5.16
19 1 1.67
20 2 3.18
21 0 0
22 2 4.19
X 17 11.39
Y 0 0
Total 177 5.29±2.90
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