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Full length Article
Association of SNPs with Rheumatoid Arthritis Susceptibility
Shakeel Safdar*1, Mahmood Kayani1, Hina Iqbal1, and Sohail Safdar2
1
Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
2
Ahlia University, Bahrain
*For
correspondence: [email protected]
ABSTRACT
Rheumatoid Arthritis is one of the types of Arthritis in which the joints swell up. It has been
found that there are approximately 340 genes involved in causing the Rheumatoid Arthritis.
Moreover, it has also been observed that SNPs of these genes are associated with the disease. In this
study, four genes were analyzed for their possible association with Rheumatoid Arthritis using
bioinformatics tools. The results showed how SNPs are associated with this disease. Some of the
SNPs brought some structural changes in particular proteins while others SNPs produced no change
in the structure. SNPs that made the proteins abnormal and brought some structural changes in the
protein can be considered as SNPs associated with Rheumatoid Arthritis, as by change of normal
amino acid, the orientations of proteins can be disturbed and protein are not involved in specific
pathways thus being pathogenic. In some cases, the structural change leads to disruption of the
protein structure while sometimes, the structure remains unchanged i.e. due to synonymous
mutations. This is due to a reason that sometimes, due to SNPs the abnormal amino acid is coded,
which can either disrupt or mutate the whole structure. This fact can be supported by an evidence
that the hydrophobic and hydrophilic amino acids provide specific orientations to proteins and
whenever some polar amino acid is replaced by non-polar amino acid or some non-polar amino acid
is replaced by polar amino acid or some cyclic amino acid is replaced by non-cyclic amino acid, the
abnormality occurs, which can lead to disruption of protein structure making it abnormal. In this
study, the changes in protein structure by SNPs are observed and such SNPs are predicted to be
involved in causing Rheumatoid Arthritis.
Keywords: Rheumatoid Arthritis, SNPs, Mutation, Genes, Proteins, Nucleotides, MIA, CST3,
CTLA4, HMGB1
INTRODUCTION
infections and includes joint pain. The suffix -
The name Rheumatoid Arthritis is derived from
oid means "resembling", i.e. resembling
the Greek word rheumatos, means "flowing"
rheumatic fever. Arthr means "joint" and the
which initially gave rise to the term "rheumatic
suffix
fever", an illness that can follow throat
inflammation". Thus Rheumatoid Arthritis is a
-itis,
a
"condition
involving
To cite this paper: S a f d a r , S , K a y a n i , M , I q b a l , H . , & S a f d a r , S . ( 2 0 1 7 ) . A s s o c i a t i o n o f S N P s w i t h R h e u m a t o i d A r t h r i t i s
Sus ceptibilit y). The International J our nal Of Innov ative Res ear ch In Biosc iences , 1(1), 56 -67.
Safdar et al. (2017), 1(1), 56-67
form of joint inflammation, which resembles
The cause of Rheumatoid Arthritis is a very
rheumatic fever. Rheumatoid Arthritis appears
active area of worldwide research. Genes and
to have been described in paintings more than
their SNPs have been found to be involved in
a century before the first detailed medical
Rheumatoid Arthritis, and in addition, the
description of the condition in 1800 (Azaola,
scientists believe that the environmental
2013).
factors
Rheumatoid arthritis (RA) is believed to be an
Rheumatoid Arthritis. Moreover, it is also
‘inflammatory autoimmune disorder’, in which
believed that smoking tobacco also increases
the joints get severely affected (Voll et al.,
the severity of Rheumatoid Arthritis (Baka et
2008). It is such a condition, in which the
al., 2009).
inflammation and joint pains destructure the
Many studies have reported the involvement of
joints, hence resulting in restriction of joint
genes and SNPs in the pathogenesis of the
mobility (William et al., 2004). RA is a
disease, some of which are mentioned here
systemic disease, often affecting ‘extra-
(Mattyasouszky et al., 2006; Kochil et al.,
articular tissues’ all over the body, which
2004; Arlehaq et al., 2005; Vazgiourakis et al.,
includes blood vessels, skin, lungs, heart, and
2007; Shimada et al., 2007). The above-
the
2010).
mentioned studies signify the involvement of
Rheumatoid Arthritis normally affects the
SNPs of various genes in Rheumatoid Arthritis.
joints but sometimes it affects the organs as
We have conducted experiments on MIA,
well. Normally small joints are affected but
CST3, CTLA 4 and HMGB 1 gene. About
larger joints can also be affected by the
CST3,
Rheumatoid
2010).
association of CST3 gene with Rheumatoid
Rheumatoid arthritis affects women three times
Arthritis. Gang Li (2014) mentioned in his
more than men and it can develop at any age.
research work that certain polymorphism in the
Women aged 40 to 50 years usually suffer from
nucleotides in CTLA4 gene has signified its
this disease. Moreover, Rheumatoid Arthritis
association with the Rheumatoid Arthritis.
occurs 4 times more in smokers than the non-
Earlier, Gonzalez (1994) also mentioned in his
smokers (Williams et al., 2004). The research
research that how CTLA4 is possibly linked
work in Bioinformatics made it evident that
with
certain change at genetic level is one of the
highlighted CTLA4’s association with RA in
major
Arthritis.
Chinese population. About HMGB1, Arnandis
Nucleotide
(2010) and Voll (2008) elaborated that they had
Polymorphism (SNP) is the reported to be one
found certain elevated range of HMGB1 genes
of the major causes of Rheumatoid Arthritis
in the synovial fluid samples of the patients
(Saad et al, 2016).
with Rheumatoid Arthritis (Arnandis, 2010).
muscles
causes
(Cojocaru
Arthritis
of
Significantly,
et
at.,
(Wilke,
Rheumatoid
Single
are
Gauthier
Rheumatoid
Earlier,
57
also
Bosserhoff
involved
(2012)
in
causing
signified
Arthritis.
(2002),
Lie
and
the
(2005)
then
Safdar et al. (2017), 1(1), 56-67
Yeremenko and his research team, highlighted
Polymorphism, so we used ClustalW® for
MIA gene’s association with the Rheumatoid
better sequential analysis. The next step was to
Arthritis (Bosserhoff, 2002; Yeremenko et al,
observe the replaced amino acid position in
2013).
normal protein sequence. As the SNP region
MATERIALS AND METHODS
sequences were only available in nucleotide
In this study, SNPs of Four genes were
sequence form, so they needed to be translated
analyzed namely MIA, CST3, CTLA4, and
in order to align with normal protein sequence.
HMGB1. The main objective of the project was
So for this purpose, TranSeq was applied which
to observe the structural changes in the protein
can translate nucleotide sequence into protein
structures, which are mentioned by NCBI to be
sequence. After translation, the translated SNP
involved in causing RA. However, in order to
region sequence and normal protein sequence
attain that objective, there were some minor
were aligned by using a protein alignment tool
objectives i.e. searching for SNPs using SNP
Tcoffee. The alignment sometimes gave the
database, selection of SNPs present in Exon
signified
region of DNA sequence, identification of
allingmnents were dispersed or scattered. The
mutation
of
reason was that different protein translation
synonymous from nonsynonymous SNPs and
tools translates nucleotide sequences by using
to find the particular region of SNP in gene
different frames, so shattered alignments can be
sequence.
observed in such cases. After the application of
causing
SNPs,
filteration
result,
but
sometimes
the
Various databases and bioinformatics tools
Tcoffee, the next step was towards the major
were applied in order to attain the minor
objective i.e Structural analysis through
objectives leading to major objective. The gene
structure modeling. The tools that were applied
sequences, protein sequences and the SNP
for structural analysis were Swiss Model server
region sequences were retrieved by NCBI. The
and Swiss PDB viewer. The Swiss model takes
tools were applied further to locate the exact
the input sequence of protein and models it.
SNP region in the gene and protein sequences.
The normal protein sequences were manually
The initial step was to align the gene sequence
mutated according to the mutation mentioned
with the SNP region sequence to locate the
by NCBI and then the structures were modeled.
exact SNP position in the gene sequence. The
In the case of MIA, when the 17th SNP
applied tool was ClustalW®, which is a
mutation was manually inserted in the normal
multiple sequence alignment tool. Higgins
protein sequence, the structure was not
(1988) in his research work has elaborated that
modeled which indicated towards disruption of
how multiple sequence pairwise alignments
protein structure due to the insertion of
can better initiate a gene/protein similarity
abnormal amino acid in protein sequence. The
study. As we had to do the sequence alignments
Swiss Model server models the protein
for
structure on homology basis i.e. it searches the
analyzing
the
Single
Nucleotide
58
Safdar et al. (2017), 1(1), 56-67
relevent or homolog protein in database and
structure. When the normal and the abnormal
designs the homology modeling. As the
protein structures were overlapped by using
structure was not designed in case of MIA 17th
Swiss PDB viewer, the result obtained shows
SNP, so we predict that there is no such protein
that the orientation of the protein structure has
present in protein database, which resembles
been changed. Therefore, we predict that SNP
with mutated protein having the abnormal
is RA causing SNP as it produces structural
amino acid, this means that such abnormal
change in protein.
protein does not exists normally in nature, so
The encircled area in the result obtained by the
we predicted the involved SNP as the RA
overlapping of normal and abnormal MIA
causing SNP. The remaining six proteins were
(consist of few residues of SNP region) shows
also analysed by using this Swiss Model server
the difference in the orientation of both the
but the models were produced. The final step
proteins. The 2nd analyzed gene was CST3
was the application of Swiss PDB viewer. In
which has total of 58 SNPs out of which 4
order to use this tools, the PDB files were
SNPs were selected i.e. SNP number 10, 14, 19
needed which were obtained by Swiss Model
and 20. In case of 10th SNP, the conversion was
as well as by Protein Data Bank. The Swiss
C to A and the amino acid change was Arg to
PDB veiwer modeled the structures for every
Ser at position 71. Arg is hydrophillic basic
SNP including the 17th SNP of MIA gene.
residue whereas Ser is hydrophillic polar
RESULTS AND DISCUSSION
residues which are of completely opposite
The intermediate steps including ClustalW,
natures. The result obtained by Swiss PDB
TranSeq and TCoffee were used to obtain the
viewer is mentioned in Fig 2.
minor objectives i.e. to move towards the
The fig 2 shows the change in orientations of
structural analysis. The reults that were
both the overlapped proteins. The secondly
obtained by the structural analysis are
selected SNP of CST3 was 14th number SNP in
mentioned in this paper. The first analyzed
which the polymorphism or conversion is G to
gene was MIA whose total SNPs, mentioned in
A and the amino acid change is Arg to His at
NCBI, are 20 out of which one SNP was
71 amino acid position. The polymorphism is
selected for analysis due to its occurance in
at the same position as that of SNP number 10.
Exon region. The SNP change was C to T that
Arg and His both are hydrophillic basic
results in replacing Thr to Ile at at 113th amino
residues but the change in structure was
acid position of the protein. Thr is a
observed because His has cyclic structure
hydrophillic polar residue, that is why it is
where as Arg does not have cyclic structure.
usually present on outer region of protein
The structural analysis obtained by Swiss PDB
structure but Ile is hydrophobic non-polar
viewer is shown in Fig 3.
residue which tends to move inward of protein
59
Safdar et al. (2017), 1(1), 56-67
Fig 1: Result caused by C to T polymorphism
Fig 2: Result caused by C to A polymorphism
60
Safdar et al. (2017), 1(1), 56-67
Fig 3: Result caused by G to A polymorphism
Fig 4: Result caused by C toG polymorphism
61
Safdar et al. (2017), 1(1), 56-67
Fig 5: Result caused by G to C polymorphism
Fig 6: Result caused by T to C polymorphism
62
Safdar et al. (2017), 1(1), 56-67
Fig 7: Result caused by A to G polymorphism
Fig 8: Result caused by C to T polymorphism
63
Safdar et al. (2017), 1(1), 56-67
Fig 9: Result caused by C to T polymorphism
64
Safdar et al. (2017), 1(1), 56-67
The SNP number 19 has a conversion of C to
abnormal protein structures shows the change
G and the amino acid change is Arg to Gly at
in protein orientation, mentioned in Fig 7.
amino acid position 96. Arg is hydrophillic
The fourth gene analyzed was HMGB1 which
basic residue and Gly is hydrophillic polar
has a total of 60 SNPs out of which 2 SNPs
residue. The result obtained by Swiss PDB
were selected for analysis. As the purpose was
viewer is shown in Fig 4. in which change in
to analyze the effect of polymorphism on the
protein orientation can be noticed.
protein creation, so we took only 2 SNPs out of
60. The conversion of nucleotide base and the
The fourth analyzed SNP of CST3 was 20th
amino acid change was same in both the SNPs
number SNP in which the conversion is G to C
i.e. C to T nucleotide conversion and Pro to Leu
and the amino acid change is Gly to Ala at 38th
amino acid change. The first SNP was 33rd
amino acid position. Gly is hydrophillic polar
number SNP in which the mutation was at 143rd
residue where as Ala is hydrophobic non-polar
position and in case of second analyzed SNP
residue. The overlaping of normal and
abnormal
protein
structures
i.e. SNP number 47, the mutation was at 98th
showed the
position. Pro and Leu both are hydrophobic
orientation change in protein structure.
non-polar residues but Pro has cyclic structure
The third gene analyzed was CTLA4, which
while Leu has linear, non-cyclic structure. The
has total 79 SNPs out of which 2 SNPs were
overlaping of normal and abnormal protein
selected for analysis. The first analyzed SNP
structures of both the cases i.e. Fig 8 shows the
was number 28 in which the nucleotide
33rd SNP case and Fig 9 shows 47th SNP case.
conversion was T to C and the amio acid
CONCLUSION
change was Met to Thr at amino acid position
It is evident that various genes are involved in
91. Met is a hydrophobic non-polar amino acid
Rheumatoid Arthritis and it has been observed
while Thr is a hydrophillic polar amino acid.
that various SNPs of different genes are
Thr tends to arrange itself towards outer region
of
protein
surface
while
Met
associated with Rheumatoid Arthritis. Our
being
research was based on analyzing the structural
hydrophobic residue, tends to move inward of
changes caused by the SNPs in the respective
the protein structure. When the normal and the
proteins of above-mentioned genes. In order to
abnormal protein structures were overlapped,
analyze the relationship of SNPs with
the change in protein orientation was observed
Rheumatoid Arthritis, the SNPs of four genes
which is mentioned in Fig 6.
namely MIA, CST3, CTLA4, and HMGB1
The second analyzed SNP was 37th SNP in
were analyzed and the effects of SNPs were
which the nucleotide conversion was A to G
pointed out that how SNP was changing the
and the amino acid change was Thr to Ala at
structure of a protein. The SNPs of the exon
17th amino acid position. Thr is hydrophillic
regions were only selected for the analysis
residue while Ala is hydrophobic non-polar
(excluding the SNPs of intronic region). The
residue. The overlaping of normal and
65
Safdar et al. (2017), 1(1), 56-67
Database used for the data collection was
single genetically homologous population.
NCBI. There were about 17 SNPs of the above-
Lupus. 16, 867-874.
mentioned genes which were found to be
mutation causing. These SNPs replaced the
Mattyasouszky, S., Skapeoko, A., Kalden, JR.,
normal amino acids with the mutated ones.
Lipsky, PE. And Schulze, H. (2006).
When the normal and mutated proteins were
INFGR1 Single Nucleotide Polymorphism
over lapped, the proteins were not able to
in Rheumatoid Arthritis. Arthritis Resther,
overlap properly at the point of mutation, which
8, 63.
indicates that there was a structural difference
Xu, B., Ärlehag, L., Rantapää-Dahlquist, S. B.,
caused by the SNP and this mutation can
& Lefvert, A. K. (2005). β2 Adrenoceptor
become the cause of Rheumatoid Arthritis.
gene single nucleotide polymorphisms are
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