Human TLR1 Deficiency Is Associated with Impaired Mycobacterial Signaling and Protection from Leprosy Reversal Reaction

Toll-like receptors (TLRs) are important regulators of the innate immune response to pathogens, including Mycobacterium leprae, which is recognized by TLR1/2 heterodimers. We previously identified a transmembrane domain polymorphism, TLR1_T1805G, that encodes an isoleucine to serine substitution and is associated with impaired signaling. We hypothesized that this TLR1 SNP regulates the innate immune response and susceptibility to leprosy. In HEK293 cells transfected with the 1805T or 1805G variant and stimulated with extracts of M. leprae, NF-κB activity was impaired in cells with the 1805G polymorphism. We next stimulated PBMCs from individuals with different genotypes for this SNP and found that 1805GG individuals had significantly reduced cytokine responses to both whole irradiated M. leprae and cell wall extracts. To investigate whether TLR1 variation is associated with clinical presentations of leprosy or leprosy immune reactions, we examined 933 Nepalese leprosy patients, including 238 with reversal reaction (RR), an immune reaction characterized by a Th1 T cell cytokine response. We found that the 1805G allele was associated with protection from RR with an odds ratio (OR) of 0.51 (95% CI 0.29–0.87, p = 0.01). Individuals with 1805 genotypes GG or TG also had a reduced risk of RR in comparison to genotype TT with an OR of 0.55 (95% CI 0.31–0.97, p = 0.04). To our knowledge, this is the first association of TLR1 with a Th1-mediated immune response. Our findings suggest that TLR1 deficiency influences adaptive immunity during leprosy infection to affect clinical manifestations such as nerve damage and disability

Single lesion multibacillary leprosy, a treatment enigma: a case report

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Comparative genomic and phylogeographic analysis of Mycobacterium leprae

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6 x coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38 x coverage) and NHDP63 (46 x coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy

Stroke genetics informs drug discovery and risk prediction across ancestries

Previous genome-wide association studies (GWASs) of stroke — the second leading cause of death worldwide — were conducted predominantly in populations of European ancestry1,2. Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis3, and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach4, we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry5. Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries

Baseline characteristics of leprosy subjects.

1<p>Percentages of individuals in some categories do not sum to 100 due to missing data. Not shown among all leprosy are 8 subjects with peripheral neuropathy (PN), and one with leprosy of indeterminate type (IN).</p>2<p>The four most frequent ethnic groups (from > 8 ethnic groups) are tabulated.</p>3<p>P represents exact <i>P</i> values for overall distribution of gender and age groups within the different ethnic groups and for the overall distribution of leprosy type and immune reactions among the 4 different ethnic groups.</p

Frequency of TLR1 haplotypes in reversal reaction.

1<p>Order for 6 SNP haplotypes, left to right: Trs5743563C, Ars5743565G, Trs5743592C, Trs5743595C, G743A, T1805G.</p>2<p>Order for 2 SNP haplotypes: Trs5743592C, T1805G.</p>3<p>P value represents comparison of a given haplotype with the reference haplotype TATTAT.</p

TLR1 Polymorphism Frequency in Reversal Reaction.

1<p>P value for comparison of allele frequencies by Chi-square.</p>2<p>P value for comparison of genotype frequencies calculated by Chi-square unless otherwise indicated.</p>3<p><b><i>A</i></b> denotes common allele, <b><i>a</i></b> denotes minor allele.</p>4<p>P value calculated by Fisher's Exact test due to small cell number.</p

IL-β and TNF-α production in human mononuclear cells following stimulation with <i>M. leprae</i>.

<p>Peripheral blood mononuclear cells were stimulated for 18 hours and supernatants were assayed for cytokine production by luminex multiplex bead assay. PBMCs were obtained from 11 individuals with the genotype 1805TT or 1805TG (TT/TG: dark circles) and 10 individuals with the 1805GG genotypes (GG: open circles). PBMCs stimulated with media, PAM2 at 75 ng/mL (P2), PAM3 at 75 ng/mL (P3), or LPS at 10 ng/ml (LPS), whole irradiated ML at 20 µg/ml (ML) or MLcw at 10 µg/ml (MLcw). IL-1β production following stimulation is shown in (A), and TNF-α production in (B). The mean level and standard error of the mean are depicted and were derived from averaging the responses of individual within each genotype group. The median level is depicted by a bar; *p≤0.05, **p≤0.001 by Mann–Whitney U-test.</p

IL-6 production by human primary cells following stimulation with <i>M. leprae.</i>

<p>Peripheral blood mononuclear cells were stimulated for 18 hours and supernatants were assayed for cytokine production by ELISA. PBMCs were derived from whole blood taken from 15 individuals with the genotype 1805TTor 1805TG (TT/TG: dark circles) and 13 individuals with the 1805GG genotypes (GG: open circles). (A): PBMCs stimulated in triplicate with media, PAM2 at 75 ng/ml (P2 75), PAM3 at 75 ng/ml (P3 75), or LPS at 10 ng/ml (LPS 10). (B): PBMCs stimulated in triplicate with whole irradiated ML at 20 or 100 µg/ml (ML20 or ML100) or MLcw at 2 or 10 µg/ml (MLcw 2 or MLcw10). The mean level and standard error of the mean are depicted and were derived from averaging the responses of individuals stimulated in triplicate. The median level is depicted by a bar. *P≤0.01, **P≤0.001 by Mann–Whitney U-test.</p

TLR1 Polymorphism Frequency in Different Leprosy Types.

1<p>P value for comparison of allele frequencies.</p>2<p>P value for comparison of genotype frequencies.</p>3<p><b><i>A</i></b> denotes common allele, <b><i>a</i></b> denotes minor allele.</p>4<p>Tuberculoid includes TT and BT. Lepromatous includes LL, BL, and BB.</p