GENETIC SUSCEPTIBILITY TO LEISHMANIA

PREPARED BYMOHANAD ALHAFIZ

SARRAH ALNOR

Introduction

• Leishmaniasis is a parasitic disease transmitted by the bite of sand flies.

• Found in parts of at least 88 countries including the Middle East• Three main forms of leishmaniasis

• Cutaneous: involving the skin at the site of a sandfly bite• Visceral: involving liver, spleen, and bone marrow• Mucocutaneous: involving mucous membranes of the mouth and nose after

spread from a nearby cutaneous lesion (very rare)

• There are different species ,L .major, donovani, trpica, braziliensis

• An intracellular parasite.• Celluar immune response• Exist in two form:• Promastigote• Amastigote• Diagnosis by detection of amastigotes by biopsy of

the liver or spleen confirms the diagnosis. Serology or PCR may support the diagnosis.

•

In Sudan ,

• Nilosaharan speaking Masalit population, are more susceptible

to leishmaniasis. They have:

• High rates of clinical disease.

• Their neighbors, the Hawsa tribe, have :

• low rates of clinical disease

• These observations convinced researchers that host genotype

might play an important role in susceptibility to disease, and

many researches have been done to examine this hypothesis.

Mice experiments

• Experimental work has provided the first evidence for the regulation of infection by host genetic factors.

• The results indicated a control by a single locus lsh on chromosome1 in mice

• Lsh was linked to other loci controlling susceptibility to Mycobacterium bovis and M.lepaemurium (locus Bcg) and Salmonella typhimurium (locus Ity) infections

• The fact that these pathogens reside in macrophage phagosome and neutralize macrophage anti-microbicidal activity suggested that Ity, Lsh and Bcg could be a single locus so the locus was known as(lsh,bcg,ity)

• After Positional cloning the locus was renamed as NRAMP1

• nucleotide sequence analysis showed that a single mutation (a G to A substitution at nucleotide position 783) resulted in the non-conservative replacement of Gly to Asp within NRAMP1 encoded protein.(D. Malo, K. Vogan, S. Vidal, et al).

Genome wide scan

• Study subjects are from the village of Barbar El Fugara in eastern Sudan. The population of this village comprises mostly from three tribes Hausa and Fellata and the Aringa

• To reduce genetic heterogeneity in the study sample, only Aringa were included(El-Safi et al).

• This was the first report of a successful genome scan in human leishmaniais (CL or KA)

• The study was conducted from 1995 to 2000• 63 nuclear families were included in the study

Study subject

• Thirty-eight families were studied in the first stage of this genome wide linkage scan, and the 25 remaining families were included only in the second stage

Study Stages

Stage1387

microsatellite markers

NRAMP1 gene (2q35) —5′

(CA)n, 469+14G/C,

and 1729+55del4—

22q12, 2q24, and 2q35 suggestive linkage

(P<.025)

• previous epidemiological studies suggested that KA occurring late in the outbreak developed against an immunological background different from that of the early cases (Bucheton et al).

• the analysis was also performed on KA cases that occurred at the beginning of the outbreak (1995- 1999).

Results

• LOD score was increased to 3.90 (P=10−5) at 22q12

• and were moderately increased to 1.00 at 2q35 (P=0.015) with NRAMP1 intragenic polymorphisms.

Conclusion

• The genotypic data from the 30 patients who developed KA late in the outbreak (in 1999 and 2000) contributed little to the LOD score that mapped a KA-susceptibility gene on chromosome 22q12.

• This indicates that the control of KA in subjects affected early in the epidemic may be different from that in subjects affected later.

• This view is supported by the observation that subjects affected late in the epidemic, unlike those affected earlier, were found to control infection for several years before the onset of clinical disease (Bucheton et al)

• This study shows a significant linkage of KA to chromosome 22q12 and provides suggestive linkage results at a second locus on chromosome 2q23-q24

Candidate genes

• The IL-2 receptor β chain gene (IL2RB), which is located in the 22q12 chromosomal region, is a good candidate for the control of KA.

• IL-2 is critical for T-cell proliferation and differentiation. T-cell responses are strongly suppressed in patients with KA (Carvalho et al. 1981, 1985), and high levels of circulating soluble IL-2 receptor have been observed during the acute phase of the disease (Barral-Netto et al.).

• Studies of other protozoa, such as Trypanosoma cruzi, also support the hypothesis that some parasites evade the immune system by acting on the IL-2 pathway (Majumder and Kierszenbaum 1996)..

• it is interesting to note that two other genes—N-myc interactor and signal transducing adapter molecule 2 (STAM2 ) both located in the 2q22-q23 region—code for proteins involved in the IL-2 signalling-transduction pathway.

The second genome scan

• second genome-wide scan undertaken in two(El-Rugab and Um-Salala)villages occupied by the related Masalit ethnic group in eastern Sudan

• The two villages have high rates of clinical VL.• Those two village are under founder effect and

consanguineal marriages• For 48 nuclear families the scan was

performed using 360 microsatellite

Results

• linkage on Chromosome 1 resolved into two clear village-specific peaks at 1p22 for (peak LOD score= 2.81; p=0.00016) in um-salala

• for linkage at on 1q31.3 in El-Rugab (LOD score 1.59; p=0.003)

• 6q27 provided evidence for a common susceptibility locus (LOD score 2.13; p= 0.000874) affecting both villages

Y-chromosome linkage

• Using Y-chromsome haplo type as marker• This provide positive linkage at 1p22 in Um-

Salala with E3b1 Y-chromosome haplotype LOD =5.65 (p = 0.00000017).

• El-Rugab village showed no linkage to disease at 1p22 for the two haplotypes.(A3b2 or E3b1)

• 6q27 for El-Rugab was also specific to E3b1 families peak LOD score = 3.74 (p=0.0000168),

• Um-Salala village showed no linkage to disease at the major peak at 6q27

Candidated genes at 1p22

• DR1, which encodes the down regulator of transcription which inhibits transcription by binding to the TATA box binding protein

• glomulin which is antiproliferative for T cells • Growth factor-independent 1 (GFI1), which

influences myeloid differentiation

Candidated genes at 6q27

• TBP(TATA box binding protein).• The Notch ligand delta-like 1 and proteasome

subunit beta-type 1 (PSMB1). • Proteasome function is important in degradation

of proteins by antigen-processing cells• Notch pathway instruct T cell differentiation .

Specifically, T helper 1 cells to release interferon-g which is crucial for immune control of L. donovani

SLC11a1 & VLthat is more familiar by its former designation Nramp1/NRAMP1.

Nramp1 stands for ‘‘natural resistance associated macrophage protein 1’’,

SLC11A1 STUDY

• carried on Nilo-Saharan speaking Masalit population occupy villages along the Rahad River in the heart of the endemic area in eastern Sudan.

• Using 67 nuclear families with one to six affected offspring per Nuclear families.

markersMARKER No. Of alleles

(GT)n 4

274C/T 2

469+14G/C 2

D543N 2

3’UTR TGTG 2

3’UTR CAAA 2

D2S1471 17

First stepLinkage analysis

using ALLERGO software

provides evidence for linkage between VL and markers across SLC11A1,

Results marker Lod score P-value

(GT)n 1.41 0.008

274C/T 1.40 0.008

469+14G/C 1.40 0.008

D543N 1.24 0.012

3’UTR TGTG 1.23 0.012

3’UTR CAAA 1.23 0.012

D2S1471 0.86 0.028

Extended transmission disequilibrium testing for single markers

showed significant global associations for

the 5’ markers

but not for the 3’ markers

Results Haplotypes Transmission% P-value comment

3-2-1 65 0.003 Disease associated

4-1-2 86 0.058 all transmissions from one extended pedigree.

2-1-2 25 0.005 Disease protective

• only the promoter (GT)n is known to be functional in regulating the expression of SLC11A1.

• To determine the main effects at each locus a forward stepwise logistic regression was conducted

• Overall, this stepwise analysis indicates that all of the association between SLC11A1 and the disease can be accounted for by the 469 +14 G/C polymorphism.

• promoter polymorphism (–86G/A) was located within a putative nuclear factor kappa B binding site that could be functional

• Further work can determine whether additional polymorphisms occur upstream in the promoter, which could be in linkage disequilibrium with the intron 4 polymorphism.

linkage and association with IL4 and IFNGR1and visceral leishmanaisis in Sudan

• Study was done in IEND by HS Mohamed 2003• Out come of leishmania infection depend on which T

helper cells will be activated• Th1 clearance of infection• Th2 disease• In this context, genes that regulate, or control

response to, type 1 and type 2 cytokines are good candidate susceptibility loci.

• linkage and significant allelic association between markers within the genes encoding type 2 cytokines IL4 and IL9 and underlying susceptibility to VL in Sudan.

• Genes causing a defect in the type 1 cytokine pathway. Polymorphism in the IFNGR1 gene encoding the type 1 IFNg receptor

• They examined 59 multicase families of visceral leishmaniasis (VL) with/without post Kala-azar dermal leishmaniasis from masalit group

• They used two types of markers

Markers IL-4 IL-9 INFGR1

VNTR Interon2IL-4RP2

- -

Microsatalite Interon3IL-4RP1

Interon4 interon6

Linkage Analysis Results

• Linkage was determined by using multipoint nonparametric analysis

IL-4RP2 IL-4RP1 IL-9 INFYR1

VL with PKDL

P=0.008 0.031 0.009 0.031

Vl per se P=0.002 0.013 0.034 0.056

• These data provide suggestive evidence for agene(s) at 5q23.3–q33 controlling underlying susceptibility to VL in this Sudanese population.(IL4,IL9)

• Weak evidence for linkage of INFYR1 was observed for PKDL (P=0.031), but not for VL per se.

• This data differed from that reported for the Arenga ethnic group , which failed to demonstrate linkage to markers in the region of IL4 and INFGR1. This might be due to reduced power in the smaller sample size (37) multicase families) used in that study(Bucheton, B. et al. (2003)

• To narrow down the region of interest that might carry the susceptibility allele within this type 2 cytokine gene cluster, allelic association was done

Allelic Association Analysis

• Allelic association was determined using thetransmission disequilibrium test (TDT).• The single markers IL4RP2 and IL4RP1 show

significant allelic association with VL alone, PKDL and VL per se

• No significant allelic or genotype associations were observed for IL9

• Results show association in the presence of linkage between VL and IL4 ( p = 0.008) but not IL9, suggesting that the etiological functional variant lies closer to IL4 than IL9

Case-pseudocontrol

• significant allelic associations with VL per se in the presence of linkage were observed for allele 2 at IL4RP2 (P=0.0034) and allele 4 at IL4RP1 (P=0.0007)

Step wise logestic regression

• both IL4RP1 allele 4 and IL4RP2 allele 2 contribute significantly to the association, suggesting a common disease-associated functional variant in linkage disequilibrium (LD) with a 2–4 haplotype for IL4RP2– IL4RP1.

Final results •Overall, the results are consistent with a gene in the close vicinity of the IL4 locus controlling underlying susceptibility to VL, whereas IFNGR1 is specifically related to susceptibility to PKDL

• One of the recommendation of this study was that further work is required to verify this result and to identify the disease associated mutation/polymorphism in this population.

• Another study looked for allelic association between INFYR1 and PKDL was done

PKDL

• In Sudan, more than 50% of cases successfully treated form visceral symptoms caused by Leishmania donovani go on to develop post-kala-azar dermal leishmaniasis (PKDL).

• PKDL is charachrarized by high level of INFY• Interferon-g (IFN-g) is a key T helper 1 cell cytokine. It

activates macrophages to kill parasites , exerting its effects through its receptor IFNGR1.

• There is high level of INFY which is good , so why it doesn’t exert its effect through the activation of MQ and then killing of the parasite?

• Inhibitory effect of IL10• Low level of INFyR1

• In this study they genotyped 80 trios from the Masalit ethnic group.The study aimed to determine the role of IFNγ and IFNγR1 gene polymorphisms in Susceptibility to PKDL among selected Sudanese population

Genotyping :

-470/471ddelTT

-270T/C -56T/C

ATG

+95T/C

5` 3`

SNPs within IFNγ gene promoter

 

-179G/A 

5` 

3` 

+874T/A

ATG

SNPs within IFNγR1 gene promoter

Allelic associationPedcheck

HWE test

TDT analysis

Case/pseudo control analysis

Haplotype base score test

 Gene

 SNP

 SNP transition

N. genotypedInformativeTransition

 X2

 df

 P-value

 IFN-γ

 -179 IFNγ

 G A

 75 (13)

 0.769

 1

 0.7815

 IFN-γ

 874 IFNγ

 T A

 71 (19)

 0.4737

 1

 0.4912

 IFNGR1

 -470 IFNGR1

 TT -

 81 (8)

 2.0000

 1

 0.1572

 IFNGR1

 -270 IFNGR1

 T C

 81 (14)

 4.5714

 1

 0.0325

 IFNGR1

 -56 IFNGR1

 T C

 77 (47)

 0.1915

 1

 0.6617

 IFNGR1

 +95 IFNGR1

 T C

 80 (47)

 0.5319

 1

 0.4658

TDT test for 6 SNPS within IFNG and IFNGR1.

• Conditional logistic regression showed that PKDL was associated with the common T allele at IFNGR1 -270 (P=0.045), whereas the minor C allele was associated with protection (P=0.046).

Haplotype Analysis

• Eleven haplotypes were present in the Masalit

• They looked for haplotype associations using TRANSMIT Haplotype analysis using all four markers showed (P=0.033) a significant bias in transmission of haplotype insTT T T T but not in transmission of the haplotypes insTT T C C or insTT TC T.

This suggests involvement of a functional variant in PKDL susceptibility that is in LD with the - 56 bp SNPS even though single marker analysis did not show a significant association with PKDL disease.

• Significant associations for three marker (e.g. insTT C C; insTT C T) and two marker (e.g. C C and C T for -270/-56) haplotypes were also associated with less than expected transmission of haplotypes carrying the - 270C

allele (Red one)

Conculsion

• No direct association between PKDL and either -470 or -56 bp polymorphisms.

-470,-56

• Rare -270C allele and haplotypes that contained this variant were associated with protection from PKDL,-270C

• The elevated IFNg response in PKDL does not appear to be associated with polymorphisms at the IFNg gene

INFY genes

HLA and visceral leishmanaisis

Negative results for linkage and association

Brazil by

C S Peacock 2002

Masalit in Sudanby

H.S Mohamed 2003

Arenga in Sudanby

Bucheton et al 2003