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Insights into the possible role of IFNG and IFNGR1 in Kala-azar and Post Kala-azar Dermal Leishmaniasis in Sudanese patients.

Salih MA, Fakiola M, Abdelraheem MH, Younis BM, Musa AM, ElHassan AM, Blackwell JM, Ibrahim ME, Mohamed HS - BMC Infect. Dis. (2014)

Bottom Line: Variable expression of IFNG was detected in lymph node aspirates of VL patients before treatment, with a marked reduction (P = 0.006) in expression following treatment.IFNGR1 expression was also variable in lymph node aspirates from VL patients, with no significant reduction in expression with treatment.Identification of novel potentially functional rare variants at IFNGR1 makes an important general contribution to knowledge of rare variants of potential relevance in this Sudanese population.

View Article: PubMed Central - PubMed

Affiliation: Institute of Endemic Disease, University of Khartoum, P. O. Box 102, Khartoum, Sudan. msalih56@hotmail.com.

ABSTRACT

Background: Little is known about the parasite/host factors that lead to Post Kala-azar Dermal Leishmaniasis (PKDL) in some visceral leishmaniasis (VL) patients after drug-cure. Studies in Sudan provide evidence for association between polymorphisms in the gene (IFNGR1) encoding the alpha chain of interferon-γ receptor type I and risk of PKDL. This study aimed to identify putative functional polymorphisms in the IFNGR1 gene, and to determine whether differences in expression of interferon-γ (IFNG) and IFNGR1 at the RNA level are associated with pathogenesis of VL and/or PKDL in Sudan.

Methods: Sanger sequencing was used to re-sequence 841 bp of upstream, exon1 and intron1 of the IFNGR1 gene in DNA from 30 PKDL patients. LAGAN and SYNPLOT bioinformatics tools were used to compare human, chimpanzee and dog sequences to identify conserved noncoding sequences carrying putative regulatory elements. The relative expression of IFNG and IFNGR1 in paired pre- and post-treatment RNA samples from the lymph nodes of 24 VL patients, and in RNA samples from skin biopsies of 19 PKDL patients, was measured using real time PCR. Pre- versus post-treatment expression was evaluated statistically using the nonparametric Wilcoxon matched pairs signed-rank test.

Results: Ten variants were identified in the 841 bp of sequence, four of which are novel polymorphisms at -77A/G, +10 C/T, +18C/T and +91G/T relative to the IFNGR1 initiation site. A cluster of conserved non-coding sequences with putative regulatory variants was identified in the distal promoter of IFNGR1. Variable expression of IFNG was detected in lymph node aspirates of VL patients before treatment, with a marked reduction (P = 0.006) in expression following treatment. IFNGR1 expression was also variable in lymph node aspirates from VL patients, with no significant reduction in expression with treatment. IFNG expression was undetectable in the skin biopsies of PKDL cases, while IFNGR1 expression was also uniformly low.

Conclusions: Uniformly low expression of IFN and IFNGR1 in PKDL skin biopsies could explain parasite persistence and is consistent with prior demonstration of genetic association with IFNGR1 polymorphisms. Identification of novel potentially functional rare variants at IFNGR1 makes an important general contribution to knowledge of rare variants of potential relevance in this Sudanese population.

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Related in: MedlinePlus

Graphical representation of theIFNGR1upstream multiple alignment generated in SynPlot. The sequences of each species are shown as lines interrupted by spaces corresponding to the gaps inserted for optimum global alignment. The horizontal axis represents the distance from the start of the alignment, and the vertical axis represents the percentage identity score generated by SynPlot (scale, 0%–100% sequence identity across all species). Peak regions that correspond to conserved noncoding sequences, as opposed to coding sequences or repeat features, are numbered. Other features are colour coded according to the key.
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Fig1: Graphical representation of theIFNGR1upstream multiple alignment generated in SynPlot. The sequences of each species are shown as lines interrupted by spaces corresponding to the gaps inserted for optimum global alignment. The horizontal axis represents the distance from the start of the alignment, and the vertical axis represents the percentage identity score generated by SynPlot (scale, 0%–100% sequence identity across all species). Peak regions that correspond to conserved noncoding sequences, as opposed to coding sequences or repeat features, are numbered. Other features are colour coded according to the key.

Mentions: Sequencing of the immediate promoter region identified 4 novel variants in the Sudanese population. However, minor allele frequencies were low making it unlikely that these variants contributed on their own to risk and protective haplotypes for PKDL. Therefore in silico methods were employed to interrogate an extended region up to 3.5 kb upstream of the initiation start site of the IFNGR1 gene. The strategy was to: (i) identify regions of conserved sequence (CNS) that are more likely to carry regulatory elements; (ii) identify TFBS within these CNS using predictive software tools; and (iii) determine whether known SNPs located within CNS cause loss or gain of relevant transcription factor binding activity. To identify CNSs, comparative sequence analysis was performed between human, chimpanzee, and dog for ~3.5 kb upstream region of IFNGR1 gene. A number of CNSs were identified across the region based on the criteria of 70% similarity over at least 100 bp of ungapped alignment [21,22]. Within this region we identified 3 CNSs in the intergenic region upstream of the IFNGR1 gene (Figure 1) which we interrogated for public domain SNP information. In total we found information on 21 SNPs across the 3.5 kb regions upstream of the IFNGR1 initiation site, 13 of which fell within CNSs as annotated on Figure 1. No known SNPs were located within peak 1. Seven SNPs were identified in peaks 2 and 3, as presented in Table 3. Two out of the ten SNPs that were detected in the sequence analysis (Table 2) are located within the CNS labelled peak 4 which encompasses the 5′UTR (Figure 1). IFNGR1 rs41401746 (−470 bp TT/-- INDEL) upstream of IFNGR1 is positioned within peak 4, which showed 81% identity over 182 nucleotides across human, chimpanzee, and dog sequences. The rs141836145 (−270 bp T/C) SNP, which was found to be associated with PKDL in our previous study [7], is also located within peak 4. Four known SNPs (rs55961762, rs55640745, rs56300633, and rs17181653), also located within peak 4, were not polymorphic in the 30 PKDL samples that were sequenced (data not shown).Figure 1


Insights into the possible role of IFNG and IFNGR1 in Kala-azar and Post Kala-azar Dermal Leishmaniasis in Sudanese patients.

Salih MA, Fakiola M, Abdelraheem MH, Younis BM, Musa AM, ElHassan AM, Blackwell JM, Ibrahim ME, Mohamed HS - BMC Infect. Dis. (2014)

Graphical representation of theIFNGR1upstream multiple alignment generated in SynPlot. The sequences of each species are shown as lines interrupted by spaces corresponding to the gaps inserted for optimum global alignment. The horizontal axis represents the distance from the start of the alignment, and the vertical axis represents the percentage identity score generated by SynPlot (scale, 0%–100% sequence identity across all species). Peak regions that correspond to conserved noncoding sequences, as opposed to coding sequences or repeat features, are numbered. Other features are colour coded according to the key.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265480&req=5

Fig1: Graphical representation of theIFNGR1upstream multiple alignment generated in SynPlot. The sequences of each species are shown as lines interrupted by spaces corresponding to the gaps inserted for optimum global alignment. The horizontal axis represents the distance from the start of the alignment, and the vertical axis represents the percentage identity score generated by SynPlot (scale, 0%–100% sequence identity across all species). Peak regions that correspond to conserved noncoding sequences, as opposed to coding sequences or repeat features, are numbered. Other features are colour coded according to the key.
Mentions: Sequencing of the immediate promoter region identified 4 novel variants in the Sudanese population. However, minor allele frequencies were low making it unlikely that these variants contributed on their own to risk and protective haplotypes for PKDL. Therefore in silico methods were employed to interrogate an extended region up to 3.5 kb upstream of the initiation start site of the IFNGR1 gene. The strategy was to: (i) identify regions of conserved sequence (CNS) that are more likely to carry regulatory elements; (ii) identify TFBS within these CNS using predictive software tools; and (iii) determine whether known SNPs located within CNS cause loss or gain of relevant transcription factor binding activity. To identify CNSs, comparative sequence analysis was performed between human, chimpanzee, and dog for ~3.5 kb upstream region of IFNGR1 gene. A number of CNSs were identified across the region based on the criteria of 70% similarity over at least 100 bp of ungapped alignment [21,22]. Within this region we identified 3 CNSs in the intergenic region upstream of the IFNGR1 gene (Figure 1) which we interrogated for public domain SNP information. In total we found information on 21 SNPs across the 3.5 kb regions upstream of the IFNGR1 initiation site, 13 of which fell within CNSs as annotated on Figure 1. No known SNPs were located within peak 1. Seven SNPs were identified in peaks 2 and 3, as presented in Table 3. Two out of the ten SNPs that were detected in the sequence analysis (Table 2) are located within the CNS labelled peak 4 which encompasses the 5′UTR (Figure 1). IFNGR1 rs41401746 (−470 bp TT/-- INDEL) upstream of IFNGR1 is positioned within peak 4, which showed 81% identity over 182 nucleotides across human, chimpanzee, and dog sequences. The rs141836145 (−270 bp T/C) SNP, which was found to be associated with PKDL in our previous study [7], is also located within peak 4. Four known SNPs (rs55961762, rs55640745, rs56300633, and rs17181653), also located within peak 4, were not polymorphic in the 30 PKDL samples that were sequenced (data not shown).Figure 1

Bottom Line: Variable expression of IFNG was detected in lymph node aspirates of VL patients before treatment, with a marked reduction (P = 0.006) in expression following treatment.IFNGR1 expression was also variable in lymph node aspirates from VL patients, with no significant reduction in expression with treatment.Identification of novel potentially functional rare variants at IFNGR1 makes an important general contribution to knowledge of rare variants of potential relevance in this Sudanese population.

View Article: PubMed Central - PubMed

Affiliation: Institute of Endemic Disease, University of Khartoum, P. O. Box 102, Khartoum, Sudan. msalih56@hotmail.com.

ABSTRACT

Background: Little is known about the parasite/host factors that lead to Post Kala-azar Dermal Leishmaniasis (PKDL) in some visceral leishmaniasis (VL) patients after drug-cure. Studies in Sudan provide evidence for association between polymorphisms in the gene (IFNGR1) encoding the alpha chain of interferon-γ receptor type I and risk of PKDL. This study aimed to identify putative functional polymorphisms in the IFNGR1 gene, and to determine whether differences in expression of interferon-γ (IFNG) and IFNGR1 at the RNA level are associated with pathogenesis of VL and/or PKDL in Sudan.

Methods: Sanger sequencing was used to re-sequence 841 bp of upstream, exon1 and intron1 of the IFNGR1 gene in DNA from 30 PKDL patients. LAGAN and SYNPLOT bioinformatics tools were used to compare human, chimpanzee and dog sequences to identify conserved noncoding sequences carrying putative regulatory elements. The relative expression of IFNG and IFNGR1 in paired pre- and post-treatment RNA samples from the lymph nodes of 24 VL patients, and in RNA samples from skin biopsies of 19 PKDL patients, was measured using real time PCR. Pre- versus post-treatment expression was evaluated statistically using the nonparametric Wilcoxon matched pairs signed-rank test.

Results: Ten variants were identified in the 841 bp of sequence, four of which are novel polymorphisms at -77A/G, +10 C/T, +18C/T and +91G/T relative to the IFNGR1 initiation site. A cluster of conserved non-coding sequences with putative regulatory variants was identified in the distal promoter of IFNGR1. Variable expression of IFNG was detected in lymph node aspirates of VL patients before treatment, with a marked reduction (P = 0.006) in expression following treatment. IFNGR1 expression was also variable in lymph node aspirates from VL patients, with no significant reduction in expression with treatment. IFNG expression was undetectable in the skin biopsies of PKDL cases, while IFNGR1 expression was also uniformly low.

Conclusions: Uniformly low expression of IFN and IFNGR1 in PKDL skin biopsies could explain parasite persistence and is consistent with prior demonstration of genetic association with IFNGR1 polymorphisms. Identification of novel potentially functional rare variants at IFNGR1 makes an important general contribution to knowledge of rare variants of potential relevance in this Sudanese population.

Show MeSH
Related in: MedlinePlus