Accepted Manuscript

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.

Figure 1

Figure 2

Figure 3

Figure 4

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