6. association of tap1 and tap2 polymorphisms in...

151 TAP polymorphisms in leprosy

6. ASSOCIATION OF TAP1 AND TAP2 POLYMORPHISMS

IN LEPROSY

INTRODUCTION

Leprosy caused by Mycobacterium leprae primarily affects peripheral

nerves and skin. Disease presents as spectrum and is governed by the

host immune response. In an endemic pocket, on exposure to M.

leprae, majority of the population do not develop overt clinical disease

indicating the involvement of host immunity in disease development

(Martelli et al 2000, Siddiqui et al 2001, Kumar et al 2004, Ogunbiyi

et al 2004, Meima et al 2004, WHO Weekly Epidemiological Report,

2007). Thus, outcome of leprosy is in part due to host genes that

controls the initial infection and the host immune response (Scollard

et al 2006). A substantial body of evidence suggests that multi

factorial genetic factors control susceptibility of humans to infection

by M. leprae (Jamieson et al 2004, Mira et al 2004, Alter et al 2008,

Da Silva et al 2009). Evidence for the role of host genetics in leprosy or

the type of disease manifestation was obtained from epidemiological

segregation and twin studies (Ishii et al 1993). Candidate gene

analysis and genome wide studies have implicated various genes and

regions on chromosomes in susceptibility or resistance towards the

infection (Miller et al 2004, Zhang et al 2009) One of the first

candidate gene studied in leprosy was Transporter Associated with

antigen presentation (TAP) (Rajalingam et al 1997). Two linkage


studies have associated chromosome 6pq21 with leprosy susceptibility

(Abel et al 2008; Mira et al 2003).

TAP is a member of the family of ABC transporters that play a key role

in the processing and presentation of endogenous antigen to CD8+ T

cells by MHC class I pathway. In contrast, MHC class II restricted

antigens are generated by endosomal proteases and presented to

CD4+ T cells. Yet, exogenous antigens can be channeled via the

endogenous pathway where antigen presentation is mediated by MHC

class I molecules (Robinson et al 2002). Thus, the ER could be a site

for loading newly synthesized MHC class II molecules and it may play

a dual role in both endogenous and exogenous antigen processing

(Tewari et al 2005).

TAP is made up of TAP1 and TAP2 polypeptides which consist of 11

and 9 exons respectively. The significance of TAP proteins for antigen

presentation to CD8+ T cells has been demonstrated both in cell lines

and in knockout mice. Restoration of TAP1 and TAP2 in cell lines

deficient in these molecules completely restored the ability of these

cells to present antigen to T lymphocytes (Van Kaer et al 1992). It is

possible that polymorphisms in the TAP gene can influence the

antigen peptide selection, transport process and alter immune

response regulation.


Over 13 polymorphic sites have been reported in the TAP1 gene

(Colona et al 1992, Powis et al 1993, Shi et al 1997, Tang et al 2001,

Lajoie et al 2003). The most commonly reported polymorphic residues

are at positions 333 and 637 (TAP1), 565 and 665 (TAP2). Because of

their endogenous antigen presentation and location on MHC, TAP

genes have been known to be important candidate genes in

autoimmune diseases and polymorphisms have been implicated in

multiple sclerosis, rheumatoid arthritis, allergic rhinitis and Insulin

dependent diabetes mellitus (Middleton et al 1994, Singal et al 1994,

Cailit- Zuchman et al 1995, Ishihara et al 1996, Foley et al 1999, Kim

et al 2007, Feng et al 2009).

Mycobacterial infectious disease like leprosy and tuberculosis are

known to associated with HLA-DR2 and its subtypes HLA DRB 1501

and 1502 ( Mehra et al 1992, Mehra et al 1995, Rajalingam et al

1996) . The TAP gene is located between HLA-DP and HLA-DQ of

chromosome 6 which has been implicated in susceptibility to leprosy

(Mira et al 2003, Zhang et al 2009). There is dearth in information of

TAP polymorphisms in South Indian population, hence we embarked

upon this study to elucidate the association of TAP1 and TAP2

polymorphisms in leprosy.


6.1 Methodology

6.1.1 Study Subjects:

Leprosy patients (n=100) reporting to two LEPRA projects: BPHRC,

HYLEP, and Sivananda Rehabilitation home were enrolled in the study

after obtaining informed written consent. All patients had slit skin

smear examination and skin lesion was evaluated by histopathology.

Out of the total 100 patients, 71 were classified as lepromatous and

29 as tuberculoid. Control subjects were endemic healthy individuals

who belonged to the similar socio-economic status and ethnic

background. None of them had any family history of leprosy,

tuberculosis or other major diseases. The study was approved by the

Institutional Ethical Committee (IEC).

6.1.2 DNA Extraction:

DNA was extracted from the biopsies using Qiagen DNeasy kit

according to the manufacturer’s instructions as mentioned in

materials and methods (Cat No 69506). DNA thus obtained was

subjected to tetra primer Amplification Refractory Mutation System

(ARMS) PCR amplification using previously published primers (Powis

et al 1993) for the TAP1 and TAP2 gene as described in chapter

materials and methods (3.12)


6.1.3 DNA amplification using ARMS-PCR:

TAP1 and TAP2 genes were amplified using a tetra primer

Amplification Refraction Mutation System-PCR (ARMS-PCR) as

described earlier (Newton et al 1989) as described in materials and

methods (3.15). The protocol was further standardized with

modifications in annealing temperatures. Polymorphic residues at

codon 333 and 637 for TAP1, 565 and 665 for TAP2 were analyzed. A

set of four primers were used for each polymorphic site. Sequencing

using ABI 3130 for a few representative samples for the detection of

polymorphic residues was outsourced from a local facility.

6.1.4 Statistical analysis:

All statistical analyses were performed with SPSS and Graph Pad

Prism ((version 5) IL, USA). A chi-square test was used to compare the

distribution of TAP1 and TAP2 genotypes between control and leprosy

groups. Odds ratios (OR) with a 95% confidence interval (CI) were

calculated using the Fisher’s exact chi-square test. Differences were

considered significant if p<0.05. Hardy-Weinberg law was used to

analyze all the possible genotype frequencies and according to the

classical method to assess if the system is in genetic equilibrium and

if there was a significant excess of homozygosity. Haplotype analysis

were performed using the Haploview v4.2 software (Barrett et al, 2005)


6.2 Results

6.2.1 TAP1 333 Isoleucine to Valine polymorphism:

The heterozygous genotype (Isoleucine (A)/Valine (G)) was positively

associated with leprosy (71%) when compared with controls (46%)

(p<0.0005). The frequency of AA genotype was significantly low in

patients (27%) when compared to controls (50%) presenting negative

association of this genotype with the disease group (p<0.001).

Frequency of GG homozygous genotype was observed to be similar in

patients and controls with 2% and 4% respectively. G allele was

positively associated with the disease when compared A allele (Table

6.1). Significance was not observed across leprosy spectrum for TAP1

333 (Table 6.2). The genotype frequencies of controls were not found

to be in Hardy-Weinberg equilibrium.

PCR products when resolved on 2% agarose gel electrophoresis

showed internal control band of 533bp, Valine specific band of 351bp

and Isoleucine specific band of 241bp. Hence the presence of two

bands of 351 and 241 basepairs in a single lane along with the 533 bp

control indicates Isoleucine/Valine heterozygote, while a 351 bp or

241 bp amplicon indicates homozygosity for Valine or Isoleucine

respectively (Figure 6.1).


Figure 6.1: ARMS PCR for TAP1 gene position 333

A representative agarose gel (2%) picture showing amplification

products for TAP1 gene at position 333.

Lanes 1,3, 5,7 and 8 - heterozygous for Isoleucine/Valine (AG).

Lanes 2, 4 and 6 - homozygous for Isoleucine (AA).

Lane 5 - homozygous for Valine (GG).

Lane 9 - 100 bp molecular marker.

Internal control (533bp) is common for all PCR products.


Fig 6.2: Chromatograms showing genotype of TAP1333

A

B

C

Partial chromatograms of TAP1 gene at position 333 showing peaks

A: Homozygous for Isoleucine ATC; B: Homozygous for Valine

C: Heterozygous for Isoleucine/Valine as indicated by overlap of A

and G peaks.


Table 6.1: Distribution of TAP1 (333) genotypes and Allele in leprosy

and control group

Table 6.2: Distribution of TAP1 (333) genotypes and alleles across

leprosy spectrum.

Using Fisher’s exact test the distribution of TAP1 333 genotypes and

alleles was compared. % represents the percentage frequency of

genotype; *p-value<0.05 was considered significant; OR: Odds Ratio;

CI: Confidence intervals.

Genotype

/ Alleles

Amino

acid

Leprosy

(100)

n(%)

Control

(100)

n(%)

Odds

ratio

(OR)

95% CI p value

TAP1333

Genotype

AA Iso/Iso 27(27) 50(50) 0.36 0.20-0.66 0.001*

AG Iso/Val 71(71) 46(46) 2.87 1.60-5.15 0.0005*

GG Val/Val 2(2) 4(4) 0.48 0.08-2.73 NS

Allele

A Iso 125 146 0.61 0.40-0.94 0.032*

G Val 75 54 1.62 1.0-2.47 0.032*

Genotype

/ Alleles

Amino

acid

BL+LL

(71)

n(%)

BT

(29)

n(%)

Odds

ratio

(OR)

95% CI p value

TAP1 333

Genotype

AA Iso/Iso 21(27.6) 8(27.6) 1.1 0.38-3.34 0.99

AG Iso/Val 48(71.6) 21(72.4) 0.79 0.26-2.23 0.82

GG Val/Val 2 0

Allele

A Iso 88 37 0.99 0.52-1.89 0.99

G Val 50 21 0.99 0.52-1.89 0.99


6.2.2 TAP1 637 to Aspartate Glycine (A to G) polymorphism

A significantly higher frequency of the heterozygous genotype (Aspartic

Acid (A)/Glycine (G)) (54%) was observed in patients when compared

to controls (34%). The frequency of AA genotype was significantly low

in patients (45%) when compared to controls (60%). On the other

hand the frequency of GG homozygous was observed to be similar in

patients (1%) and controls (6%) respectively. G allele was positively

associated with the disease when compared A allele (Table 6.3). No

significance was observed across leprosy spectrum for TAP1 637

(Table 6.4). The genotype frequencies of controls were not found to be

in Hardy-Weinberg equilibrium.


showed control band of 429bp, Aspartic Acid specific band of 380bp

and Glycine specific band of 180bp. Hence the presence of two bands

of 380 and 180 basepairs in a single lane along with the 429 bp

control indicates Aspartic Acid/glycine heterozygote, while a 380 bp or

180 bp amplicon indicates homozygosity for Aspartic Acid or Glycine

respectively (Figure 6.3).


Figure 6.3: ARMS PCR for TAP1 gene position 637

A representative agarose gel (2%) picture showing amplification

products for TAP1 gene at position 637.

Lanes 3, 4 and 10 - heterozygous for Aspartic Acid/Glycine (AG)

Lanes 2,5,6,7,8,9 and 10 - homozygous for Aspartic Acid (AA)

Lane 9 is a 100 bp molecular marker



Fig 6.4: Chromatograms showing genotype of TAP1637

A

B

C

Partial chromatograms of TAP1 at position 637 showing peaks

A: Homozygous for Aspartic Acid ; B:Homozygous for Glycine

C: Heterozygous for Aspsartic Acid/Glycine as indicated by overlap of

A and G peaks


Table 6.3: Distribution of TAP1 (637) genotypes and alleles in leprosy

patients and control group

Table 6.4: Distribution of TAP1 (637) genotype and alleles across

leprosy spectrum

Using Fisher’s exact test the distribution of TAP1 637 genotypes and

alleles was compared. % represents the percentage frequency of



Genotype

/ Alleles

Amino

acid

Leprosy

(100)

n(%)

Control

(100)

n(%)

Odds ratio

(OR)

95% CI p value

TAP1 637

Genotype

AA Asp/Asp 45(45) 60(60) 0.54 0.31-0.95 0.047*

AG Asp/Gly 54(54) 34(34) 2.27 1.28-4.03 0.006*

GG Gly/Gly 1(1) 6(6) 0.15 0.01-1.33 0.11

Allele

A Asp 144 154 0.68 0.40-1.08 0.13

G Gly 56 46 1.47 0.92-2.37 0.13

Genotype /

Alleles

Amino

acid

BL+LL

(71)

n(%)

BT (29)

n(%)

Odds

ratio

(OR)

95% CI p value

TAP1 637

Genotype

AA Asp/Asp 36(50.7) 10(34.5) 1.9 0.79-4.78 0.18

AG Asp/Gly 34(47.9) 19(65.5) 0.48 0.19-1.18 0.12

GG Gly/Gly 1(1.4) 0

Allele

A Asp 106 39 1.4 0.73-2.79 0.3

G Gly 36 19 0.69 0.35-1.35 0.3


6.2.3 TAP2 565 Alanine to Threonine (G to A) polymorphism:

The frequency of homozygous genotype (Alanine (G)/Alanine (G)) was

similar in patients (95%) and controls (97%). Frequency of

heterozygous GA genotype had no significant difference between

patient (3%) and control (4%) (Table 6.5). Significant association was

not seen for TAP2 565 between the leprosy spectrum (Table 6.6). The

genotype frequencies did not follow Hardy-Weinberg equilibrium


showed control band of 400bp, Alanine specific band of 298bp and

Threonine specific band of 161bp. Hence the presence of two bands of

298 and 161 base pairs in a single lane along with the 400 bp control

indicates Alanine/Threonine (GA) heterozygote, while a 298 bp or 161

bp amplicon indicates homozygosity for Alanine(GG) or Threonine(AA)

respectively(Figure 6.5)


Fig 6.5. ARMS PCR for TAP1 gene position 565

A representative agarose gel (2%) image showing amplified products

for TAP2 gene for position 565.

Lane 1 - 100bp molecular marker.

Lanes 2,3,4,5 and 6 - homozygous for Alanine/Alanine(GG).

Lane 7 - heterozygous for Alanine/Threonine (GA).



Table 6.5 : Genotype and Allele distribution of TAP2 (565) gene

polymorphism.

Table 6.6: Genotype and Allele distribution of TAP2(565) gene across

leprosy spectrum

Using the Fisher’s exact test the distribution of TAP2 565 genotypes

and alleles were compared. % represents the percentage frequency of



Genotype /

Alleles

Amino

acid

Leprosy

100

n(%)

Control

(100)

n(%)

Odds ratio

(OR)95% CI p value

TAP2 565

Genotype

GG Ala/Ala 97(97) 95(95) 1.7 0.39-7.32 0.72

GA Ala/Thr 3(3) 4(4) 0.58 0.13-2.52 0.72

AA Thr/Thr 0 1(1) 0.33 0.01-8.20 1

Allele

G Ala 197 195 1.68 0.39-7.14 0.72

A Thr 3 5 0.59 0.14-2.51 0.72

Genotype /

Alleles

Amino

acid

BL+LL

(71)

n(%)

BT (29)

n(%)

Odds

ratio

(OR)

95% CI p value

TAP2 565

Genotype

GG Ala/Ala 70(98.59) 27(93.10) 5.18 0.45-59.60 0.2

GA Ala/Thr 1(1.40) 2(6.89) 0.19 0.01-2.21 0.2

AA Thr/Thr 0 0 - - -

Allele

G Ala 142 56 5.07 0.45-57.08 0.2

A Thr 1 2 0.19 0.09-2.21 0.2


6.2.4 TAP2 665 Threonine to Alanine (A to G) polymorphism:

There was no significant difference between leprosy patients and

control group for TAP2 665. Frequency of heterozygous AG genotype

in patients (58%) was similar to controls (66%) and homozygous AA in

patients (30%) and controls (40%). Alleles A and G were equally

distributed in the leprosy patient and control groups (Table 6.7 and

6.8) Significant association was not seen for TAP2 665 between the

leprosy spectrums or when lepromatous or tuberculoid patients were

compared with the control group. The genotype frequencies were

found to be in Hardy-Weinberg equilibrium.


showed control band of 408bp, Threonine specific band of 326bp and

Alanine specific band of 141bp. Hence the presence of two bands of

326 and 141 base pairs in a single lane along with the 408 bp control

indicates Threonine /Alanine heterozygote, while a 326 bp or 141 bp

amplicon indicates homozygosity for Threonine or Alanine respectively

(Fig.6.6)


Fig 6.6 Agarose gel (2%) showing ARMS PCR showing amplification of

TAP2 665

Fig 6.6 : A representative 2% agarose gel electrophoresis showing

amplification products for TAP2 gene at position 665.Lane 1, 2,3 and

5 were heterozygous for Alanine/Threonine (TA) ; Lane 4 and 6 were

homozygous for threonine (AA) and Lane7 is 100bp molecular marker;

Internal control (408bp) is common for all PCR products


Table 6.7: Genotype and Allele distribution of TAP2 (665) gene

polymorphism.

Genotype / Alleles

Amino acid

Leprosy

(100) n(%)

Control (100) n(%)

Odds ratio (OR)

95% CI p value

TAP2 665

Genotype

AA Thr/Thr 40(40) 30(30) 1.56 0.86-2.79 0.18

AG Thr/Ala 58(58) 66(66) 0.71 0.40-1.26 0.3

GG Ala/Ala 2(2) 4(4) 0.48 0.08-2.73 0.68

Allele

A Thr 138 126 1.3 0.86-1.97 0.24

G Ala 62 74 0.76 0.50-1.15 0.24

Table 6.8: Percent distribution of genotype and allele for TAP2(665)

across leprosy spectrum

Using the Fisher’s exact test the distribution of TAP2 665 genotypes

and alleles were compared. % represents the percentage frequency of



Genotype /

Alleles

Amino

acid

BL+LL

(71)

n(%)

BT

(29)

n(%)

Odds

ratio

(OR)

95% CI p value

TAP2 665

Genotype

AA Thr/Thr 28(39.43) 12(41.38) 0.92 0.38-2.22 1

AG Thr/Ala 41(57.74) 17(23.94) 0.96 0.40-2.32 1

GG Ala/Ala 2(2.81) 0 -

Allele

A Thr 97 41 0.89 0.45-1.74 0.86

G Ala 45 17 1.18 0.60-2.30 0.86


Table 6.9: Association of TAP1 haplotype with infection outcome.

Table 6.10: Association of TAP2 haplotype with infection outcome

Figures in parenthesis represent the sample size in each cohort

Using the Fisher’s exact test the frequency of haplotypes in control

and patient groups for the TAP2 665 were compared. % represents the

percentage frequency of genotype; *p-value<0.05 was considered

significant; OR: Odds Ratio; CI: Confidence intervals.

TAP1 Control (100) Patients (100)

Haplotypes

(333/637) n % n % OR CI(95%) p value

AA

(Iso/Asp)126 63 126 63 1 0.66-1.5 1

GG

(Val/Gly)55 27 26 13 0.39 0.23-0.66 0.0004

GA

(Val/Asp)18 9 28 14 1.64 0.87-3.08 0.1576

AG

(Iso/Gly)1 1 20 10 20 2.65-150 0.0001

TAP2 Control (100) Patients (100)

Haplotypes

(565/665) n % n % OR CI(95%) p value

GA

(Ala/Thr)124 62 135 67 1.27 0.84-1.92 0.2953

GG

(Ala/Ala)71 35 62 31 0.81 0.53-1.23 0.3939

AA

(Thr/Thr)5 3 3 2 0.59 0.14-2.52 0.2462


Table 6.11: Association of combined haplotypes of TAP1 and TAP2 to

infection outcome

Using the Fisher’s exact test the frequency of haplotypes in control

and patient groups for the TAP gene (positions 333,637,565,665) were

compared. *p-value<0.05 was considered significant.

Control (100) Patients (100)

Haplotypes

TAP1/TAP2

333/637/565/665

n % n % OR CI p- value

AAGA

(Iso/Asp/Ala/Thr)63 31.5 63 31.5 1 0.65-1.52 1

AAAG

(Iso/Asp/Ala/Ala)63 31.5 59 29.5 0.84 0.55-1.29 0.5169

GGGA

(Val/Gly/Ala/Thr) 24 12.0 52 26.0 2.57 1.51-4.38 0.0005*

GAGA

(Val/Asp/Ala/Thr)21 10.5 19 9.5 0.89 0.46-1.72 0.8679

AGGA

(Iso/Gly/Ala/Thr)16 8.0 2 1.0 0.11 0.02-0.51 0.001*

AAAA

(Iso/Asp/Thr/Thr)1 0.5 2 1.0 2.01 0.18-22.36 1

GGGG

(Val/Gly/Ala/Ala)2 1.0 2 1.0 1 0.14-7.17 1

AAAG

(Iso/Asp/Thr/Ala0 0.0 1 0.5 3.01 0.12-74.52 1

GAAA

(Val/Asp/Thr/Thr)4 2.0 0 0.0 0.11 0.005-2.037 0.1231

GAGG

(Val/Asp/Ala/Ala)2 1.0 0 0.0 0.19 0.001-4.15 0.4982

AGGG

(Iso/Gly/Ala/Ala)4 2.0 0 0.0 0.18 0.005-2.032 0.12


6.2.5 Haplotyping:

Haplotype frequency of TAP1 gene (333 and 637 position):

Results from our study suggest a positive association of TAP1

haplotypes at 333 and 637 position with leprosy. For TAP1 gene we

observed four haplotypes in both the groups. Haplotype AA was the

most frequent (63%), followed by GG, GA and AG. The TAP1 SNP

haplotype GG had a significantly higher frequency in controls when

compared to patients (p<0.0005)(Table 6.9). TAP1 haplotype AG was

significantly high in patients when compared to controls indicating a

positive association with the disease (p<0.0001), the frequency of

haplotypes AG was very low in control group (1%).

Haplotype frequency of TAP2 gene (565 and 665 position):

For TAP2 SNP three haplotypes were observed (Table 6.10). The

frequency of GA was high in both patients (67%) and controls (62%).

TAP2 SNP haplotype GG occurred with almost equal frequency in

control (31%) and patient (35%) groups. The frequency of TAP2 SNP

haplotype AA was quite low in both patient (2%) and control (3%)

groups. TAP2 haplotype AG was not detected in either of the group.

When TAP2 gene was considered at position 565 and 665, none of the

observed haplotypes were significantly associated with leprosy.


Haplotype frequency of TAP1 and TAP2 gene (333,637,565

and 665 position):

For TAP1-TAP2 SNP, 11 haplotypes were observed, the most

commonly observed was AAGA (Iso-Asp-Ala-Thr) (Table 6.11). This

haplotype was equally distributed in patient and control group

showing a high frequency (31.5%), followed by AAGG, GGAA and

GAGA. A significantly high frequency of haplotype GGGA (Val-Gly-Thr-

Ala)(p<0.0005) was seen in patients when compared to controls, thus

indicating a positive association of this haplotype with the disease. On

the other hand significantly high frequency of haplotype with

combination AGGA (p<0.001) was observed in controls when

compared to the patient group, indicating a negative association of

this haplotype with the disease.


6.3 DISCUSSION:

HLA class I molecules plays a crucial role in mammalian adaptive

immune system by presenting antigen peptides on the cell surface.

These peptides are actively transported across the endoplasmic

reticulum by transporters associated with antigen presentation

(TAP).Hence polymorphism in these genes may affect the selection

process that recognizes the antigen. One of our studies reported the

influence of TAP polymorphism on HIV-TB co-infection. The HIV

positive individuals with GG (Val/Val) at TAP1 333 and GA (Gly/Asp)

at TAP1 637 positions were at higher risk of developing TB co-infection

(Sharada et al 2011). M.leprae the causative agent of leprosy and

M.tuberculosis both belong to the mycobacterial family and the only

study has reported the association of TAP polymorphism in North

Indian tuberculoid leprosy patients (Rajalingam et al 1997). Our study

investigated the importance of TAP gene polymorphism in

susceptibility towards leprosy.

Findings from our study revealed that the heterozygous genotype AG

of TAP1 333 and TAP1 637 may confer susceptibility to leprosy and

the homozygous genotype AA protection from the disease. Genotyping

analysis findings in our study were consistent with those reported on

allergic rhinitis, ankylosing spondylitis, and cancer, dengue

hemorrhagic fever (Kim et al 2007, Soundravally et al 2007, Feng et al

2009, Einstein et al 2009). A low frequency of homozygous genotype


AA in patients was observed in this study indicating its protective role

in leprosy. The association of this genotype with protection has also

been reported in dengue (Soundravally et al 2007). However, another

study revealed a high frequency of homozygous AA genotype in on in

Polish and UK patients affected with sarcoidosis (Foley et al 1999).

The AG genotype indicating the presence of both Isoleucine and valine

at position 333 (codon) of TAP1 gene might affect peptide transport

thereby affecting the downstream processes of antigen presentation

and immune responses, which may contribute to the development of

leprosy. Thus in our population, the AG genotype at TAP1 333 seems

to be a risk factor for the disease. This is reiterated by the fact that

subjects with homozygous genotype AA seem to be resistant from

developing the disease.

Our data revealed that for TAP1 637, Aspartic acid/Glycine

heterozygote (AG genotype) was responsible for susceptibility to

leprosy making this genotype a risk factor for the disease. These

findings corroborate to those reported in cystic echinococcosis,

pneumonitis where AG genotype was reported to be a risk factor for

the disease (Galvez et al 2008, Kiper et al 2010). Interestingly in our

population the homozygous genotype (AA) Aspartic Acid was found to

be protective factor. These findings are similar to those reported in

dengue where AA homozygous genotype is protective (Soundarvally et

al 2007). This could be true for the infectious diseases, since this

genotype is reported to be associated with risk of developing atopic


dermatitis, an allergic disorder in the Chinese population (Kuwata et

al 1995).

Our results suggest that there is no influence of TAP2 polymorphic

residues at positions 565 and 665 on leprosy. Majority of the patient

and control groups were expressing homozygous GG genotype coding

for Alanine/Alanine at position 565, which was in agreement with

other diseases such as sarcoidosis and ankylosing spondilytis (Foley

et al 1999, Kim et al 2007, Feng et al 2009). Genotype AA coding for

Threonine was not detected in either the patient or control groups and

this has been reported in dengue hemorrhagic fever in an Indian

population (Soundarvally et al 2007) This finding is similar to that

seen in ankylosing spondylitis where a very low frequency of AA

genotype is reported in patient group( Feng et al 2009). At TAP2

position 665 a very low frequency of AA genotype was observed in

both, patient and control group. However a positive association of

TAP2 665 genotype AA has been reported in inflammatory rheumatoid

arthritis in Finnish population (Tuokko et al 1998)

A high frequency of TAP1 SNP rare haplotype AG was observed in our

population. This finding parallels to that reported in autoimmune

disease Systemic lupus erythematous in Columbian patients (Correa

et al 2003). Another study reports the association of TAP1 SNP

haplotype AG with juvenile myoclonic epilepsy (Layouni et al 2010).

Interestingly the rare TAP1 SNP haplotype AG was found to influence


the susceptibility to leprosy. Furthermore TAP1-TAP2 SNP haplotype

GGGA was observed to be positively associated with leprosy pointing

towards the association of this haplotype with susceptibility towards

leprosy. Conversely haplotype AGGA would possibly confer protection

from the disease.

TAP2 SNP haplotype GG was equally distributed in patient and control

group. However a study from North India reports a positive

association of TAP2B haplotype GGG at position 379/565/665 in

leprosy patients positive for HLA-DR15 (Rajalingam et al 1997). A

discrepancy in the haplotype distribution between the two studies can

be explained by a number of factors. Firstly ethnic variation might

influence the genetic regulation and related polymorphisms in

individual disease. Secondly relatively small number of patients

enrolled in that study (N=50) when compared to our study. Yet

another reason could be enrollment of only polar tuberculoid patients

in the previous study. Therefore it could be postulated that the

harboring TAP1 SNP Iso-333-Gly-637 and TAP1-TAP2 SNP and Val-

Gly-Ala-Thr haplotype combination for TAP could probably alter

antigen presentation making an exposed individual susceptible to

leprosy. Our findings also suggest that the haplotype GGGA may

cause susceptibility to leprosy and conversely haplotype AGGA was

observed to be protective.


Our results provide genetic evidence that polymorphism in the TAP1

gene influences the susceptibility to leprosy in our population. One

explanation is that the microbial peptides may not be presented or

bound efficiently to MHC class I thereby leading to a detrimental or

minimal immune response leading to increased leprosy susceptibility.

Though the findings presented here are admittedly incomplete due to

lack of HLA-DR typing, further studies on HLA polymorphisms can

reveal whether or not TAP alleles are in linkage disequilibrium with

HLA in our population. More studies will be required to understand

contribution of these polymorphisms towards susceptibility to leprosy.

Future studies focusing on the functional aspect involving TAP

expression in transcription and protein level may shed light on the

association of these polymorphisms in leprosy.

In conclusion, in our population heterozygous genotype AG indicating

the presence of both Isoleucine and Valine at codon 333 of TAP1 and

Aspartic Acid and Glycine at position of codon 637 of TAP1 might

affect peptide transport and contribute to the development of leprosy.

The results from the study also indicate the association of haplotype

GGGA(Val-Gly-Ala-Thr) with susceptibility to and AGGA(Iso-Gly-Thr-

Ala) with protection from leprosy Therefore, TAP1 may be an

important factor in the pathogenesis of leprosy.


Summary

TAP1 at position 333

Heterozygous genotype AG may be risk factor

Homozygous AA may be protective

TAP1 at position 637

Homozygous genotypes AG may be risk factor.

Homozygous genotype AA may be protective.

TAP1 SNP haplotype,

AG may confer susceptibility to leprosy

GG may protect from leprosy

TAP1-TAP2 SNP Haplotype ,

GGGA may confer susceptibility to leprosy

AGGA may protect from leprosy

TAP2 565 and TAP2 665 have no influence on leprosy

No influence of TAP1 and TAP2 polymorphic residues with

leprosy spectrum.

6. association of tap1 and tap2 polymorphisms in...

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