SLC11A1 polymorphisms and host susceptibility to cutaneous leishmaniasis in Pakistan
Sophie et al. Parasites & Vectors
SLC11A1 polymorphisms and host susceptibility to cutaneous leishmaniasis in Pakistan
Mariam Sophie 0
Abdul Hameed 2
Akhtar Muneer 1
Azam J. Samdani 3
Saima Saleem 0
Abid Azhar 0
0 Karachi Institute of Biotechnology & Genetic Engineering (KIBGE), University of Karachi , Karachi 75270 , Pakistan
1 Kuwait Teaching Hospital , Abdara Chowk, University Road, Peshawar , Pakistan
2 Institute of Biomedical and Genetic Engineering (IBGE) , 24-Mauve Area, G- 9/1, Islamabad , Pakistan
3 National Medical Centre, A-5/A, National Highway, Phase 1, Defence Housing Authority , Karachi , Pakistan
Background: The vector-borne cutaneous leishmaniasis (CL) is endemic in several regions of Pakistan mainly affecting poor populations. Host genetic factors, particularly SLC11A1 (solute carrier transmembrane protein) within macrophages, play a crucial role in disease pathology and susceptibility. Association of SLC11A1 with cutaneous leishmaniasis, a neglected tropical disease, is not well established. Inconsistencies have been observed within different populations worldwide with respect to genetic susceptibility. This study was designed to investigate genetic variation(s) in SLC11A1 and to assess possible association with cutaneous leishmaniasis in Pakistan. Results: Eight polymorphisms (rs2276631, rs3731864, rs2290708, rs2695342, rs201565523, rs17215556, rs17235409, rs17235416) were genotyped across SLC11A1 in 274 patients and 119 healthy controls. Six polymorphisms were studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing. Two single nucleotide polymorphisms were analyzed with newly designed semi-nested PCR assays. Case-control analysis showed no association between selected polymorphisms in SLC11A1 and cutaneous leishmaniasis. No significant difference was observed in the distribution of alleles between leishmaniasis patients and healthy individuals. Strong pairwise linkage disequilibrium was observed between rs2276631 and rs2290708 (r2 = 64); and rs17235409 and rs17235416 (r2 = 78). Conclusions: This study shows that genetic variations in the candidate gene SLC11A1 do not affect susceptibility to cutaneous leishmaniasis in the sample population from Pakistan.
Cutaneous leishmaniasis; SLC11A1; Single nucleotide polymorphism; Genetic susceptibility; Pakistan
Leishmaniasis is a group of diseases with diverse clinical
outcomes that range from self-healing ulcerative lesions
on the skin (cutaneous leishmaniasis) to fatal visceral
infection. This vector-borne tropical disease is caused by
the digenetic parasitic protozoa, Leishmania, which
exists as either flagellated promastigotes in Phlebotomus
sand fly vectors or non-flagellated amastigotes in
phagocytic cells. Disease manifestation depends upon the
encounter between the invading protozoa and host
organism leading to either susceptibility or resistance to
the infection [1, 2]. There are over 20 different
Leishmania species and more than 90 sand fly species
responsible for leishmaniasis and parasite transmission,
respectively. According to World Health Organization
statistics, leishmaniasis is endemic in 98 countries,
endangering 350 million people. It has an incidence of
1.3 million cases with an estimated mortality rate of
20,000 to 30,000 per year [3, 4].
In Pakistan, the visceral form of leishmaniasis is
mainly restricted to the Azad Jammu Kashmir and
Abbottabad regions in the north [5, 6]. The major
burden lies in the form of cutaneous leishmaniasis which
is reported from all parts of the country, particularly
Balochistan and Khyber Pakhtunkhwa Provinces  with
a significant proportion found in children aged 14 years
or less . The influx of refugees from Afghanistan
along the western border of Pakistan is considered as
one of the contributing factors responsible for the growing
number of cases in this region .
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Host genetics, in addition to the infecting Leishmania
species, parasite load and environmental factors, play a
crucial role in determining the type and severity of the
disease . Genome wide association studies have
identified solute carrier family 11 member a1 (SLC11A1)
gene as a strong candidate which assists in intracellular
pathogen control . SLC11A1 gene spans 12 kb in
length comprising 15 exons. These encode a 550 amino
acid protein with 10–12 predicted transmembrane
domains . It is localized to the phagosome
membrane and is involved in the transport of divalent cations
. During an intracellular infection, SLC11A1
transports essential elements (Mn2+, Fe2+, Co2+) vital for the
survival of the parasite, from the phagolysosome into the
cytosol and hence starving and restricting their growth
. Nucleotide analysis of SLC11A1 gene in inbred
mice strains revealed a single non-synonymous amino
acid substitution of glycine to aspartic acid (Gly169Asp).
Mice with this mutation were unable to produce a
functional protein which made them susceptible to
intracellular parasites . This non-conservative mutation has not
yet been identified in the human homologue SLC11A1.
Restriction and subsequent resolution of intracellular
parasites following phagocytosis by macrophages has
made SLC11A1 a strong candidate for predisposition to
different infectious diseases like tuberculosis and leprosy
[16, 17]. Genetic analysis and sequencing have identified
multiple genetic polymorphisms within the human
homologue SLC11A1 . However, these genetic
variations when studied with respect to susceptibility to
intracellular Leishmania protozoa reveal an inconsistent
pattern across different regions of the world [18–25].
Therefore, the current study was designed to determine
and analyze the genetic variation(s) in SLC11A1 gene
and investigate if these polymorphism(s) are associated
with cutaneous leishmaniasis in Pakistan.
Samples for this study were collected with informed
consent over the course of three years (2010 to 2012).
Subjects included 274 clinically diagnosed leishmaniasis
patients presenting to local hospitals of Karachi (Jinnah
Postgraduate Medical Centre and Sindh Institute of Skin
Diseases) and Peshawar (Kuwait Teaching Hospital).
Diagnosis was based on the direct microscopic visualization of
stained Leishmania amastigotes from lesion exudates.
Controls comprised a total of 119 healthy contacts
exposed to the same environment as the patients. Patients
from both genders and all ages were included in the study.
Blood samples (5 ml) were collected using acid citrate
dextrose (ACD) vacutainers to prevent coagulation.
Genomic DNA was extracted from peripheral blood
leukocytes by standard phenol-chloroform method
. Concentration of isolated DNA was determined
by spectrophotometric analysis (Analytik Jena, Jena,
Germany). Genomic DNA concentrations of 50 ng/μl
were prepared of all samples for genotyping analysis
and stored at -20 °C till further processing.
Primer design and polymerase chain reaction
Whole gene sequence of SLC11A1 was retrieved from
Ensembl database. Intron specific primers were designed
which allowed the amplification of whole or multiple
exons, depending on their size using the online software
Primer3 . Polymerase chain reaction (PCR) was
optimized for each primer set including annealing
temperature and PCR master mix concentrations. A
typical PCR reaction consisted of 30 μl reaction mixture
containing 1× PCR buffer, 1.5–3 mM MgCl2, 200 μM
deoxynucleotide triphosphates (dNTPs) each, 0.4 μM of
each forward and reverse primer and 1–3 units of Taq
polymerase (Thermo Scientific, Waltham, USA). PCR
cycle conditions included an initial denaturation step at
94 °C for 4 min followed by 35 cycles consisting of:
denaturation at 94 °C for 30 s, annealing at 55–58 °C
(specific for each primer set) for 35 s and elongation at
72 °C for 30 s, followed by a final elongation step at 72 °C
for 7 min (ThermoHybaid, Needham, USA). Five
microliters of PCR product was loaded onto 2% agarose gel
stained with ethidium bromide (0.5 μg/ml) for
electrophoresis. The PCR products were visualized under
ultraviolet (UV) illumination using Gel-Documentation
System (Bio-Rad, Hercules, USA).
Preliminary sequence analyses of at least 25 PCR
samples of each amplified exon was carried out. Prior to
sequencing, PCR products were purified by AccuPrep
PCR Purification Kit (Bioneer, Daejeon, Korea) using a
binding column tube. The purified products were
assessed on 2% agarose gel to confirm a successful
purification reaction. These were then sequenced using ABI
3730XL DNA Analyzer (Applied Biosystems, Waltham,
USA). Sequenced products were later aligned and
analyzed using the Molecular Evolutionary Genetics Analysis
(MEGA) software .
Sequenced products were scanned for DNA sequence
variations including substitutions, insertions or deletions.
Eight nucleotide variations were identified. These included
two silent substitutions; one in exon 3 (274C/T) and the
other in exon 9 (825A/G); a substitution in intron 5
(57718G/A) and intron 7 (639 + 22C/T); 3 missense mutations
(A318V in exon 9, V443A in exon 13, D543N in exon 15)
and a four base pair TGTG deletion (1729 + 55del4) in the
3′ untranslated region (3′UTR). Based on these
polymorphisms, restriction fragment length polymorphism (RFLP)
experiments were designed.
Restriction fragment length polymorphism (RFLP)
RFLP was performed for six of the above mentioned
single nucleotide polymorphisms (SNPs) for verification
of these SNPs in the remaining sample population
(Additional file 1: Table S1). These included rs2276631
(274C/T) in exon 3, rs3731864 (577-18G/A) in intron 5,
rs2695342 (825A/G) in exon 9, rs201565523 (A318V) in
exon 9, rs17235409 (D543N) in exon 15 and rs17235416
(1729 + 55del4) in 3′UTR. Restriction enzymes for each
SNP were selected using NEBcutter V2.0 . A total
volume of 30 μl reaction containing 10 μl of PCR product,
2 μl of 10× buffer and 1 μl restriction enzyme was
prepared. PCR products were digested overnight at 37 °C
in a water bath according to manufacturer’s instructions
(Fermentas, Waltham, USA). The obtained restriction
digests were resolved on 2.5% agarose gel by electrophoresis.
The ethidium bromide stained gels were visualized under
Direct genotyping of the two SNPs, rs2290708 (639 +
22C/T) and rs17215556 (V443A) in intron 7 and exon
13, respectively, was performed by designing two
seminested PCR assays, one for each SNP. Allele specific
primers were designed using PRIMER1 .
Semi-nested PCR for the SNP 639 + 22C/T in intron 7
was performed with a total volume of 15 μl reaction.
The reaction contained 200 ng of genomic DNA, 0.4 μM
of each forward outer primer (5′-CTG CGG AAG CTG
GAA GCT TTT TTT GGA C-3′) and T-allele specific
reverse inner primer (5′-GAG GAA TGA TCT TGG
GAG GTC CAC AGT TGA-3′), 0.2 μM of reverse outer
primer (5′-CAC ATA CTG CAG GGA GGA GCC TGG
TCA G-3′), 200 μM dNTPs each, 1.2 mM MgCl2, 1×
PCR buffer (400 mM KCl and 100 mM Tris-HCl,
pH 9.0) and 1 unit of Taq DNA polymerase (Bioneer).
Amplification was performed using Multi Block System
0.2S (ThermoHybaid) with the following conditions:
initial denaturation at 94 °C for 4 min, followed by 35 cycles
comprising denaturation at 94 °C for 30 s, annealing at
56 °C for 35 s and extension at 72 °C for 30 s. The last
cycle was followed by a final extension at 72 °C for
Similarly, semi-nested PCR for the SNP V443A in
exon 13 was performed in a total reaction of 15 μl
containing 200 ng genomic DNA, 1× PCR buffer, 1.5
units Taq polymerase (Thermo Scientific), 1.2 mM
MgCl2, 200 μM dNTPs each, 0.2 μM forward outer
primer (5′-CAG TTG AGC TCA CAC CCA CAC A-3′),
0.1 μM reverse outer primer (5′-ACC TCT CTC AGC
CTC TGT GGC T-3′) and 0.2 μM C-allele specific
reverse inner primer (5′-AAC GTG AGG ATG GGC ATC
A touch down semi-nested PCR approach was adopted
for the SNP V443A. PCR conditions included an initial
denaturation step at 94 °C for 4 min. The first 15 cycles
out of 35 cycles were run at the following conditions:
94 °C for 30 s for denaturation, 58 °C for 35 s for
annealing and 72 °C for 30 s for elongation. The cycling
conditions for the remaining 20 cycles included: denaturation
at 94 °C for 30 s, annealing at 55 °C for 35 s and
elongation at 72 °C for 30 s. The PCR reaction ended with a
final elongation at 72 °C for 7 min.
The PCR products from both semi-nested PCR assays
were run on ethidium bromide stained 2.5% agarose gel
and bands were observed by means of a gel
documentation system (Bio-Rad).
Genotypic and minor allele frequencies (MAF) were
calculated for each SNP among cases and controls.
Statistical tests including Chi-square test and odds ratio
(OR) with 95% confidence interval were performed for
association analysis of SLC11A1 gene with cutaneous
leishmaniasis using PLINK v1.07 software under the null
hypothesis of “no association” . Pairwise linkage
disequilibrium plots of the SNPs were created using
Haploview 4.2 . Power calculations for the genetic
study was undertaken following the guidelines
elaborated by Sham and Purcell . P-values less than 0.05
were considered statistically significant.
Nucleotide sequences of purified PCR samples were
analyzed by means of multiple sequence alignment with
MEGA software. These sequences were compared with
the retrieved SLC11A1 gene sequence from Ensembl
database. Alignment revealed eight nucleotide
polymorphisms spread over exon 3, intron 5, intron 7, exon 9,
exon 13, exon 15 and 3′UTR regions, respectively, some
of which had been described previously [18, 20]. The
newly designed primers which covered whole exons did
not detect any new functional polymorphisms in the
following coding regions: exon 4, 5, 6, 7, 10, 11 and 12.
The non-conservative glycine to aspartic acid
substitution formerly reported in mice was also not detected.
The total genetic variants identified in SLC11A1 are
described in Table 1.
The SNPs identified through initial sequence analysis
either created or destroyed a restriction site. Identification
Table 1 SLC11A1 polymorphisms
aCharacters in bold indicate the nucleotide change reported. SNP identity retrieved from dbSNP Short Genetic Variations
bAlleles identified in the sample population
c + = insertion of TGTG; − = deletion of TGTG
of polymorphisms in the remaining number of samples
in the given population was thus carried out by restriction
fragment length polymorphism analysis for the SNPs
rs2276631 (274C/T) in exon 3, rs3731864 (577-18G/A) in
intron 5, rs2695342 (825A/G) and rs201565523 (A318V)
in exon 9, rs17235409 (D543N) in exon 15 and rs172
35416 (1729 + 55del4) in 3′ UTR as described by Liu et al.
. The alleles of each polymorphism presented a
distinct pattern of restriction digests with agarose gel
electrophoresis after treatment with restriction enzymes.
The two newly-designed semi-nested PCR assays for the
direct identification of the SNPs in intron 7 and exon 13
produced two bands with agarose gel electrophoresis; a
larger fragment produced by outer primers and one
smaller fragment corresponding to the specific allele.
Semi-nested PCR for rs2290708 (639 + 22C/T) gave a
band at 239 bp with the outer primers for all samples. A
second band of 117 bp was observed in only those
samples which were T-allele positive. Similar results were
observed for rs17215556 (V443A) polymorphism specific
semi-nested PCR assay. A larger band at 306 bp with the
outer primers was observed for each and every sample.
While a second smaller band at 143 bp specific for the
rarer C-allele was observed only in those samples that
carried the C-allele.
Table 2 lists all the possible genotypes for each
polymorphism and summarizes the genotypic and MAF
distribution among cases and controls for all genetic
variations. In addition, MAF reported from other
populations are also listed for comparison. All SNPs were in
Hardy-Weinberg equilibrium for controls. The
polymorphisms rs3731864 (577-18G/A), rs2695342 (825A/G)
and rs201565523 (A318V) were monomorphic
exhibiting only GG, GG and CC genotype, respectively. Two
genotypes, CC and CT, were observed for the SNP
rs2276631 (274C/T) with the genotypic frequencies
exhibiting no significant difference between cases and
controls. Variations in exon 13 (rs17215556) and exon
15 (rs17235409) displayed similar results with only two
genotypes present in the sample population. TT and CT
genotypes were identified for rs17215556 while
genotypes GG and GA for rs17235409, respectively.
Genotyping for the SNP rs2290708 (639 + 22C/T) in intron 7
detected all three genotypes (CC, CT, and TT) in both
cases and controls. Homozygous 4 base pair insertion
(TGTG+/TGTG+) at 3′ UTR (rs17235416) was more
common among cases and controls as compared to
homozygous deletion (TGTG-/TGTG-). Additionally,
the heterozygous genotype (TGTG+/TGTG-) was only
observed in cases and none in controls.
Association of SLC11A1 and CL
This study included 274 Leishmania infected cases and
119 healthy controls exposed to the same environment.
Eight SNPs were genotyped, out of which 3 were
monomorphic. The remaining five SNPs were tested for
association with cutaneous leishmaniasis using PLINK, a
genomic association testing toolset. Table 3 shows the
frequency distribution of the minor alleles among cases
and controls. Single locus association test confirmed that
the null hypothesis of “no association” with cutaneous
leishmaniasis holds true for all markers tested (Table 3).
The five polymorphic SNPs (274C/T, 639 + 22C/T,
V443A, D543N and 1729 + 55del4) were further
analyzed for the non-random association of their alleles
through the pairwise linkage disequilibrium plot that
was generated by Haploview 4.2 and is shown in Fig. 1.
The plot shows pairwise D′ and r2 scores of linkage
disequilibrium (LD) between each SNP of SLC11A1 with
respect to cutaneous leishmaniasis. LD scores between
rs2276631 (274C/T) and rs2290708 (639 + 22C/T) was
r2 = 64. The missense mutation rs17235409 in exon 15
(D543N) and the insertion/deletion polymorphism
rs17235416 at 3′UTR (1729 + 55del4) appear to be in
Polymorphism Region Genotype Cases (%) Controls (%) A1a A2b MAFc
strong linkage disequilibrium (r2 = 78) whereas the
results for the other SNP pairs were either not conclusive
or strongly indicated recombination.
Genetic factors play a crucial role in host susceptibility or
resistance to infectious diseases. Variations within the
SLC11A1 gene were assessed in this study with respect to
cutaneous leishmaniasis in Pakistan which has not been
reported before from this region. A total of eight
polymorphisms present in SLC11A1 gene were analyzed and their
possible role in disease association and linkage was tested.
Six of these genetic variations (rs2276631, rs2290708,
rs3731864, rs201565523, rs17235409 and rs17235416)
have been analyzed before in various populations with
regard to leishmaniasis. In addition to these six SNPs,
two polymorphisms, rs2695342 (825A/G) in exon 9 and
rs17215556 (V443A) in exon 13, not investigated
previously for their role in cutaneous leishmaniasis, were
also included. The SNP rs2695342 (825A/G) in exon 9
alters the codon GCA to GCG. The resultant mutation
is synonymous with no change in the amino acid
alanine. Only the GG genotype was observed in all samples
(cases and controls) unanimously while the genotypes
GA and AA were absent from the population. This SNP
(rs2695342) lies in the cytoplasmic loop region between
transmembrane 6 and transmembrane 7 . Similarly,
other monomorphic polymorphisms identified were in
intron 5 (577-18G/A) and in exon 9 (A318V), with the
latter SNP expected to lie in the extracellular loop
region between transmembrane 7 and 8 . The presence
of a single genotype in the sample population may
suggest homogeneity of the alleles due to the common
geographical ancestry within the Pakistani population.
The genetic substitution GTG to GCG in exon 13
(rs17215556) is the second SNP whose role has not yet
Table 3 Single locus association test between SLC11A1 polymorphisms and CL
Polymorphism A1a F_Ab F_Uc A2d
been studied in Leishmania patients. This polymorphism
causes a modification in codon 443 which results in a
change in amino acid valine to alanine in the helical
structure of transmembrane 10 . Since this
conservative change results in the substitution of one
hydrophobic amino acid with another, it is not expected to
influence the protein structure. Literature survey shows
that association of this non-synonymous mutation with
pulmonary Mycobacterium avium complex revealed no
significant results .
Genotyping the SNPs rs2290708 (639 + C/T) in intron
7 and rs17215556 (V443A) in exon 13 was approached
by designing a semi-nested PCR. Allele identification
through semi-nested PCR for rs17215556 was slightly
modified by adding a touch-down step. This introduced
two annealing temperatures that improved allele specific
amplification. The semi-nested PCR assay made one step
genotyping possible therefore making it an efficient
technique. All three genotypes (CC, CT, TT) were observed
for rs2290708 (639 + C/T) in intron 7 with the rarer TT
genotype having a higher frequency in cases as
compared to controls. Rarer CC genotype for rs17215556
(V443A) in exon 13, was absent from the population.
Other polymorphisms including 274C/T, D543N and
1729 + 55del4 also displayed more than one genotype in
the sample population.
Minor allele frequencies were similar to other
populations including Caucasians, Africans and Asians except
for rs17235416 (1729 + 55del4) where the frequency for
TGTG deletion polymorphism at 3′ untranslated region
was much higher in African population (0.31) when
compared to the population from Pakistan.
Fig. 1 Linkage disequilibrium (LD) plot for the genotyped variations in SLC11A1 created by Haploview 4.2. For D′ LD plot, confidence bounds
color scheme was used: white, strong evidence of recombination; light grey, uninformative; dark grey, strong evidence of LD. r2 values are
represented as black for r2 = 1; white for r2 = 0; shades of grey indicating values within the range 0 < r2 < 1. Numbers within the squares
correspond to D′ and r2 scores for pairwise LD
Case-control analysis of these two SNPs (rs2695342
and rs17215556) in addition to the 6 genetic
variations previously studied in other populations, revealed
no evidence of association between these 8 loci of
SLC11A1 and cutaneous leishmaniasis patients from
Pakistan. No significant difference in distribution of
alleles between cases and controls was observed. The
polymorphisms studied do not suggest a direct impact
on structural or functional integrity of the
Pairwise linkage disequilibrium results showed strong
LD between the non-synonymous substitution rs2276631
and SNP rs2290708 in intron 7. The missense mutation
D543N in exon 15 and the 4 bp deletion of TGTG
nucleotides (1729 + 55del4) in 3′UTR regions towards the 3′ end
of the gene also appeared to be in strong linkage
disequilibrium indicating non-random segregation at these loci.
To date, only a few papers have addressed the question
of susceptibility to leishmaniasis with respect to the gene
SLC11A1. This lack of attention and funding for
research may possibly be because the disease burden is
restricted to poverty-stricken and under developed areas.
Studies on the association of SLC11A1 with
leishmaniasis exhibit contradictory and inconsistent results with
different patterns observed across the world. A study
conducted in Sudan reports strong linkage at the
SLC11A1 promoter region but no association between
the gene and disease . Similarly, another study in
Sudan revealed association of visceral leishmaniasis (VL)
with 5′ polymorphisms including (GT)n at the promoter,
274C/T in exon 3 and 469 + 14G/C in intron 4 whereas
3′ polymorphisms at exon 15 and 3′UTR loci did not
appear to have any association . However, in a
Moroccan population, only exon 15 and exon 8
polymorphic markers influence susceptibility to VL while 5′
end polymorphisms played no role in disease
This pattern of fluctuation continues across South
America where Brazilian patients carrying TGTG
insertion allele at 3′UTR showed association with cutaneous
leishmaniasis . When CL and VL were both studied
in Mexico, SLC11A1 polymorphisms were associated
with only VL and none with CL .
Interestingly, the results from Asian nations including
Sri Lanka and India were more consistent. The
polymorphic markers of SLC11A1 when analyzed in Sri Lanka
and India, failed to show any association with either CL or
VL, respectively [22, 23] as observed in this study of
Pakistani CL patients. This overlapping pattern of no
association among the south Asian countries may be
attributed to similar genetic makeup of their populations
with respect to the closeness in geography and large scale
migration of people particularly between India and
Pakistan at the time of independence.
However, other factors should be taken into account.
The infecting Leishmania species are not the same in
these three countries. In Pakistan, CL is highly endemic
with Leishmania tropica responsible for infection in the
northern and Leishmania major in the southern areas of
the country, respectively. Whereas in Sri Lanka, CL is
caused by Leishmania donovani. This species is the
causative agent of VL in India which is the
predominating form of the disease in the country.
A recent report on multilocus microsatellite typing of
Leishmania isolates obtained from cutaneous
leishmaniasis samples from Pakistan identified L. major
populations that were genetically different from those found in
Africa, Central Asia, Iran and Middle East. They propose
that this may be attributed to the different vector and
animal host species found in this region . Therefore,
in addition to host genetic factors, differences in the
clinical forms of leishmaniasis and invading Leishmania
species are also important elements that may affect the
variability in disease susceptibility.
There is a need for more SLC11A1 and cutaneous
leishmaniasis association studies to help understand the
cellular mechanism better. It also raises questions
whether other regulatory genes of the immune system
have a role to play in relation to SLC11A1 and
whether it is the combination of multiple genes that
is the underlying factor for leishmaniasis
susceptibility. The focus on cutaneous leishmaniasis is critical,
not only because it is one of the neglected tropical
diseases but also has an alarming rate of infecting
one person every 20 s in endemic areas . The
persisting disfiguring scars left after treatment of the
ulcers are associated with social stigma especially
among females . Co-infection of leishmaniasis
with human immunodeficiency virus (HIV) is another
major problem that has emerged. Cases of co-infection
from over 35 countries have been reported which add to
the burden of the disease .
This preliminary study was designed to investigate the
role of SLC11A1 gene in a Pakistani population infected
with cutaneous leishmaniasis which has not been
reported before from this region. It was demonstrated that
selected SLC11A1 polymorphisms did not affect
susceptibility to CL in the sample population. This work can
be further extended to incorporate other polymorphisms
of SLC11A1 and as well as other immune regulatory
genes and observe their combined result. The analysis of
host genetic factors will help understand better the
molecular mechanisms involved in mediating control and
elimination of disease parasites which may explain the
difference in susceptibility among different populations.
Additional file 1: Table S1. Genetic variants identified within SLC11A1
gene through exon specific amplification and restriction fragment length
polymorphism (PCR-RFLP) analysis. (DOCX 16 kb)
ACD: Acid citrate dextrose; CL: Cutaneous leishmaniasis; dNTPs: Deoxynucleotide
triphosphates; HIV: Human immunodeficiency virus; LD: Linkage disequilibrium;
MAF: Minor allele frequencies; MEGA: Molecular evolutionary genetics analysis;
OR: Odds ratio; PCR: Polymerase chain reaction; PCR-RFLP: Polymerase chain
reaction-restriction fragment length polymorphism; RFLP: Restriction fragment
length polymorphism; SLC11A1: Solute carrier family 11 member a1; SNP: Single
nucleotide polymorphism; UTR: Untranslated region; VL: Visceral leishmaniasis
We are thankful to Dr Muneer Soomro (Sindh Institute of Skin Diseases), staff at
Kuwait Teaching Hospital, Peshawar, and Jinnah Postgraduate Medical Centre,
Karachi, for providing us with samples.
MS was fully involved in all stages of the study, including sample and data
collection, molecular techniques (DNA isolation and amplification, RFLP
assays and semi-nested PCR), statistical analysis and writing the manuscript.
AH supervised the study methodology, participated in statistical analyses,
interpretation of data and reviewed the manuscript. AM and AJS are
dermatologists who organized and conducted sample and data collection
from Kuwait Teaching Hospital, Peshawar and Jinnah Postgraduate Medical
Center, Karachi, respectively. SS trained MS in the laboratory for DNA
isolation and amplification techniques. AA conceived and supervised the
overall conduct of the study. All authors read and approved the final
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Samples for this study were collected with informed consent from cases and
Approval for the study was provided by the Ethical Committee of the
Karachi Institute of Biotechnology and Genetic Engineering, University of
Karachi, Karachi, Pakistan.
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