1. GENETIC SUSCEPTIBILITY TO LEISHMANIA
PREPARED BY
MOHANAD ALHAFIZ
SARRAH ALNOR

2. 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

3. 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.

4. 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.

5. 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

6. 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)

7. 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).

8. 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)

9. The study was conducted from 1995 to 2000
Study subject
The study was conducted from 1995 to 2000
63 nuclear families were included in the study
Study Stages
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

10. Stage1 Stage 2 387 microsatellite markers
NRAMP1 gene (2q35) —5′(CA)n, G/C, and del4—
22q12, 2q24, and 2q35 suggestive linkage (P<.025)
Stage 2
five microsatellites 22q12 and 2q23-2q24 loci, three NRAMP1 gene polymorphisms
LOD scores in region 22q12 increased to 3.50 (P=3×10−5)
No increase in lod score 2q35,2q23-2q24

11. 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 ( ).

12. 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.

13. 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.

14. 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

15. 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.).

16. 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.

17. 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

18. Results
linkage on Chromosome 1 resolved into two clear village-specific peaks at 1p22 for (peak LOD score= 2.81; p= ) 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= ) affecting both villages

19. 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 = ).
El-Rugab village showed no linkage to disease at 1p22 for the two haplotypes.(A3b2 or E3b1)

20. 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

21. 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

22. 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

23. is a proton/divalent cation antiporter(Fe++
SLC11a1 & VL
Solute carrier family 11 member a1 (Slc11a1)
Located on chromosome 2q35
is a proton/divalent cation antiporter(Fe++
that is more familiar by its former designation Nramp1/NRAMP1.
Nramp1 stands for ‘‘natural resistance associated macrophage protein 1’’,

25. 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.

26. markers MARKER No. Of alleles (GT)n 4 274C/T 2 469+14G/C D543N
3’UTR TGTG
3’UTR CAAA
D2S1471 17

27. First step Linkage analysis using ALLERGO software
provides evidence for linkage between VL and markers across SLC11A1,

28. Results marker Lod score P-value (GT)n 1.41 0.008 274C/T 1.40
469+14G/C
D543N
1.24
0.012
3’UTR TGTG
1.23
3’UTR CAAA
D2S1471
0.86
0.028

29. Extended transmission disequilibrium testing for single markers
showed significant global associations for
the 5’ markers
but not for the 3’ markers

30. 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

31. 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

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

33. linkage and association with IL4 and IFNGR1 and 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
Th clearance of infection
Th disease
In this context, genes that regulate, or control response to, type 1 and type 2 cytokines are good candidate susceptibility loci.

34. 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

35. They used two types of markers
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
Interon2
IL-4RP2 -
Microsatalite
Interon3
IL-4RP1
Interon4
interon6

36. 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
Vl per se
P=0.002
0.013
0.034
0.056

37. These data provide suggestive evidence for agene(s) at 5q23
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)

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

39. Allelic Association Analysis
Allelic association was determined using the
transmission 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

40. 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

41. Step wise logestic regression
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

42. 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

43. 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.

44. 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

45. SNPs within IFNγ gene promoter
Genotyping :
SNPs within IFNγR1 gene promoter
-470/471
ddelTT
-270T/C
-56T/C
ATG
+95T/C 5` 3`
SNPs within IFNγ gene promoter
ATG
-179G/A 5` 3`
 +874T/A

46. Case/pseudo control analysis Haplotype base score test
Allelic association
Pedcheck
HWE test
TDT analysis
Case/pseudo control analysis
Haplotype base score test

47. TDT test for 6 SNPS within IFNG and IFNGR1.
Gene
SNP
 SNP transition
N. genotyped
Informative
Transition X2 df
P-value
IFN-γ
-179 IFNγ
G A
75 (13)
0.769 1
0.7815
874 IFNγ
T A
71 (19)
0.4737
0.4912
IFNGR1
 -470 IFNGR1 TT
81 (8)
2.0000
0.1572
 -270 IFNGR1
T C
81 (14)
4.5714
0.0325
 -56 IFNGR1
77 (47)
0.1915
0.6617
 +95 IFNGR1
80 (47)
0.5319
0.4658

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

49. 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.

50. 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.

51. Significant associations for three marker (e. g
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)

52. Conculsion
-56
-470,
No direct association between PKDL and either -470 or -56 bp polymorphisms.
-270C
Rare -270C allele and haplotypes that contained this variant were associated with protection from PKDL,
INFY genes
The elevated IFNg response in PKDL does not appear to be associated with polymorphisms at the IFNg gene

53. HLA and visceral leishmanaisis
Negative results for linkage and association
C S Peacock 2002
Brazil by
H.S Mohamed 2003
Masalit in Sudan
Bucheton et al 2003
Arenga in Sudan