A Systemic Review and Meta-analysis on Natural Resistance-associated Macrophage Protein 1 (3'-Untranslated Region) and Nucleotide-binding Oligomerization Domain-2 (rs8057341) Polymorphisms and Leprosy Susceptibility in Asian and Caucasian Populations.

The current meta-analysis aims to explore the potential correlation between natural resistance-associated macrophage protein 1 (NRAMP1) (3'-Untranslated region [3'-UTR]) and nucleotide-binding oligomerization domain-2 (NOD2 [rs8057341]) gene polymorphisms and their association with leprosy susceptibility in both Asian and Caucasian populations. Datas were retrieved from case control studies with NOD 2 and NRAMP 1 gene polymorphism associated with leprosy disease. Leprosy emerges as a particularly distinctive ailment among women on a global scale. The NRAMP1 (3'-UTR) and NOD2 (rs8057341) genetic variations play a crucial role in the progression of leprosy. A systematic review of relevant case-control studies was conducted across several databases, including ScienceDirect, PubMed, Google Scholar, and Embase. Utilizing MetaGenyo and Review Manager 5.4 Version, statistical analyses were carried out. Nine case-control studies totaling 3281 controls and 3062 leprosy patients are included in the research, with the objective of examining the potential association between NRAMP1 (3'-UTR) and NOD2 (rs8057341) gene polymorphisms and leprosy risk. The review methodology was registered in PROSPERO (ID520883). The findings reveal a robust association between NRAMP1 (3'-UTR) and NOD2 (rs8057341) gene polymorphisms and leprosy risk across various genetic models. Although the funnel plot analysis did not identify publication bias, bolstering these findings and elucidating potential gene-gene and gene-environment interactions require further comprehensive epidemiological research. This study identified a strong correlation between polymorphisms in the NOD2 (rs8057341) genes and susceptibility to leprosy across two genetic models. Further comprehensive epidemiological investigations are warranted to validate these findings and explore potential interactions between these genes and environmental factors.

Identification of potential biomarkers of leprosy: A study based on GEO datasets.

Leprosy has a high rate of cripplehood and lacks available early effective diagnosis methods for prevention and treatment, thus novel effective molecule markers are urgently required. In this study, we conducted bioinformatics analysis with leprosy and normal samples acquired from the GEO database(GSE84893, GSE74481, GSE17763, GSE16844 and GSE443). Through WGCNA analysis, 85 hub genes were screened(GS > 0.7 and MM > 0.8). Through DEG analysis, 82 up-regulated and 3 down-regulated genes were screened(|Log2FC| > 3 and FDR < 0.05). Then 49 intersection genes were considered as crucial and subjected to GO annotation, KEGG pathway and PPI analysis to determine the biological significance in the pathogenesis of leprosy. Finally, we identified a gene-pathway network, suggesting ITK, CD48, IL2RG, CCR5, FGR, JAK3, STAT1, LCK, PTPRC, CXCR4 can be used as biomarkers and these genes are active in 6 immune system pathways, including Chemokine signaling pathway, Th1 and Th2 cell differentiation, Th17 cell differentiation, T cell receptor signaling pathway, Natural killer cell mediated cytotoxicity and Leukocyte transendothelial migration. We identified 10 crucial gene markers and related important pathways that acted as essential components in the etiology of leprosy. Our study provides potential targets for diagnostic biomarkers and therapy of leprosy.

Genetic diversity of Mycobacterium leprae: Need to move towards genome-wide approaches.

ABSTRACT: Leprosy, an ancient disease, continues to be a public health concern as it remains endemic in several countries. After reaching the elimination target (1/10,000) as a public health problem in 2005 in India, around 1.2 lakh cases have been detected every year over the last decade indicating active transmission of leprosy bacillus (Mycobacterium leprae). Single-nucleotide polymorphisms (SNPs), genomic insertions/deletions and variable-number tandem repeats (VNTRs) have been identified as genetic markers for tracking M. leprae transmission. As the leprosy bacilli cannot be cultured in vitro, molecular testing of M. leprae genotypes is done by polymerase chain reaction-based sequencing which provides a practical alternative for the identification of strains as well as drug resistance-associated mutations. Whole-genome sequencing (WGS) of M. leprae directly from clinical samples has also proven to be an effective tool for identifying genetic variations which can further help refine the molecular epidemiological schemes based on SNPs and VNTRs. However, the WGS data of M. leprae strains from India are scarce, being responsible for a gross under-representation of the genetic diversity of M. leprae strains present in India and need to be addressed suitably. Molecular studies of leprosy can provide better insight into phylogeographic markers to monitor the transmission dynamics and emergence of antimicrobial resistance. An improved understanding of M. leprae transmission is essential to guide efficient leprosy control strategies. Therefore, this review compiles and discusses the current status of molecular epidemiology, genotyping and the potential of genome-wide analysis of M. leprae strains in the Indian context.

Mitochondrial variants of complex I genes associated with leprosy clinical subtypes.

Leprosy is a chronic bacterial infection mainly caused by Mycobacterium leprae that primarily affects skin and peripheral nerves. Due to its ability to absorb carbon from the host cell, the bacillus became dependent on energy production, mainly through oxidative phosphorylation. In fact, variations in genes of Complex I of oxidative phosphorylation encoded by mtDNA have been associated with several diseases in humans, including bacterial infections, which are possible influencers in the host response to leprosy. Here, we investigated the presence of variants in the mtDNA genes encoding Complex I regarding leprosy, as well as the analysis of their pathogenicity in the studied cohort. We found an association of 74 mitochondrial variants with either of the polar forms, Pole T (Borderline Tuberculoid) or Pole L (Borderline Lepromatous and Lepromatous) of leprosy. Notably, six variants were exclusively found in both clinical poles of leprosy, including m.4158A>G and m.4248T>C in MT-ND1, m.13650C>A, m.13674T>C, m.12705C>T and m.13263A>G in MT-ND5, of which there are no previous reports in the global literature. Our observations reveal a substantial number of mutations among different groups of leprosy, highlighting a diverse range of consequences associated with mutations in genes across these groups. Furthermore, we suggest that the six specific variants exclusively identified in the case group could potentially play a crucial role in leprosy susceptibility and its clinical differentiation. These variants are believed to contribute to the instability and dysregulation of oxidative phosphorylation during the infection, further emphasizing their significance.

Leprosy: treatment, prevention, immune response and gene function.

Since the leprosy cases have fallen dramatically, the incidence of leprosy has remained stable over the past years, indicating that multidrug therapy seems unable to eradicate leprosy. More seriously, the emergence of rifampicin-resistant strains also affects the effectiveness of treatment. Immunoprophylaxis was mainly carried out through vaccination with the BCG but also included vaccines such as LepVax and MiP. Meanwhile, it is well known that the infection and pathogenesis largely depend on the host's genetic background and immunity, with the onset of the disease being genetically regulated. The immune process heavily influences the clinical course of the disease. However, the impact of immune processes and genetic regulation of leprosy on pathogenesis and immunological levels is largely unknown. Therefore, we summarize the latest research progress in leprosy treatment, prevention, immunity and gene function. The comprehensive research in these areas will help elucidate the pathogenesis of leprosy and provide a basis for developing leprosy elimination strategies.

Association of CC-chemokine ligand-2 gene polymorphisms with leprosy reactions.

BACKGROUND: C-C motif chemokine ligand 2, a gene that codes for a protein involved in inflammation. Certain SNPs in the CCL2 gene have been studied for their potential associations with susceptibility to various diseases. These SNPs may affect the production and function of the CCL2 protein, which is involved in the recruitment of immune cells to the site of inflammation. Variations in CCL2 may influence the immune response to Mycobacterium leprae infection. OBJECTIVE: To investigate the association of the C-C motif chemokine ligand-2 single nucleotide polymorphisms with leprosy. METHODS: CCL2 single nucleotide polymorphisms were analyzed in a total of 975 leprosy patients and 357 healthy controls. Of those, 577 leprosy and 288 healthy controls were analyzed by PCR-RFLP for CCL2 -2518 A>G, 535 leprosy and 290 controls for CCL2 -362 G>C, 295 leprosy and 240 controls for CCL2 -2134 T>G, 325 leprosy and 288 controls for CCL2 -1549 A>T SNPs by melting curve analysis using hybridization probe chemistry and detection by fluorescence resonance energy transfer (FRET) technique in Realtime PCR. The levels of CCL2, IL-12p70, IFN-γ, TNF-α, and TGF-ß were estimated in sera samples and correlated with CCL2 genotypes. RESULTS: The frequency of the GCT (-2518 A>G, -362 G>C, -2134 T>G) haplotype is observed to be higher in leprosy patients compared to healthy controls (P = 0.04). There was no significant difference observed in genotypic frequencies between leprosy patients and healthy controls {(-2518A>G, p = 0.53), (-362 G>C, p = 0.01), (-2134 T>G, p = 0.10)}. G allele at the -2134 site is predominant in leprosy (borderline) without any reaction (8 %) compared to borderline patients with RR reactions (2.1 %) (P = 0.03). GG genotype (p = 0.008) and G allele at -2518 (p = 0.030) of the CCL 2 gene were found to be associated with patients with ENL reaction. An elevated level of serum CCL2 was observed in leprosy patients with the -2518 AA and AG genotypes (p = 0.0001). CONCLUSIONS: G allele and GG genotype at the CCL2 -2518 site are associated with a risk of ENL reactions.

MicroRNAs correlate with bacillary index and genes associated to cell death processes in leprosy.

Mycobacterium leprae infects skin and peripheral nerves causing a broad of clinical forms. MicroRNAs (miRNAs) control immune mechanisms such as apoptosis, autophagy as well as to target genes leading to abnormal proliferation, metastasis, and invasion of cells. Herein we evaluated miRNAs expression for leprosy phenotypes in biopsies obtained from patients with and without reactions. We also correlated those miRNAs with both, bacillary index (BI) and genes involved in the micobacteria elimination process. Our results show a significant increase in the miR-125a-3p expression in paucibacillary (PB) patients vs multibacillary (MB) subjects (p = 0.007) and vs reversal reactions (RR) (p = 0.005), respectively. Likewise, there was a higher expression of miR-125a-3p in patients with erythema nodosum leprosum (ENL) vs MB without reactions (p = 0.002). Furthermore, there was a positive correlation between miR-125a-3p, miR-146b-5p and miR-132-5p expression and BI in patients with RR and ENL. These miRNAS were also correlated with genes such as ATG12 (miR-125a-3p), TNFRSF10A (miR-146b-5p), PARK2, CFLAR and STX7 (miR-132-5p). All together we underpin a role for these miRNAs in leprosy pathogenesis, implicating mechanisms such as apoptosis and autophagy in skin. The miR-125a-3p might have a distinct role associated with PB phenotype and ENL in MB patients.

The role of neurotrophin polymorphisms and susceptibility to neural damage in leprosy.

OBJECTIVES: Mycobacterium leprae is able to infect Schwann cells leading to neural damage. Neurotrophins are involved in nervous system plasticity and impact neural integrity during diseases. Investigate the association between single nucleotide polymorphisms in neurotrophin genes and leprosy phenotypes, especially neural damage. DESIGN: We selected single nucleotide polymorphisms in neurotrophins or their receptors genes associated with neural disorders: rs6265 and rs11030099 of brain-derived neurotrophic factor (BDNF), rs6330 of BDNF, rs6332 in NT3 and rs2072446 of P75NTR. The association of genetic frequencies with leprosy phenotypes was investigated in a case-control study. RESULTS: An association of the BDNF single nucleotide polymorphism rs11030099 with the number of affected nerves was demonstrated. The "AA+AC" genotypes were demonstrated to be protective against nerve impairment. However, this variation does not affect BDNF serum levels. BDNF is an important factor for myelination of Schwann cells and polymorphisms in this gene can be associated with leprosy outcome. Moreover, rs11030099 is located in the binding region for micro-RNA (miRNA) 26a that could be involved in control of BDNF expression. We demonstrated different expression levels of this miRNA in polar forms of leprosy. CONCLUSION: Our findings demonstrate for the first time an association between the polymorphism rs11030099 in the BDNF gene and neural commitment in leprosy and may indicate a possible role of miRNA-26a acting synergistically to these genetic variants in neural damage development.

Identification of gene signatures and molecular mechanisms underlying the mutual exclusion between psoriasis and leprosy.

Leprosy and psoriasis rarely coexist, the specific molecular mechanisms underlying their mutual exclusion have not been extensively investigated. This study aimed to reveal the underlying mechanism responsible for the mutual exclusion between psoriasis and leprosy. We obtained leprosy and psoriasis data from ArrayExpress and GEO database. Differential expression analysis was conducted separately on the leprosy and psoriasis using DEseq2. Differentially expressed genes (DEGs) with opposite expression patterns in psoriasis and leprosy were identified, which could potentially involve in their mutual exclusion. Enrichment analysis was performed on these candidate mutually exclusive genes, and a protein-protein interaction (PPI) network was constructed to identify hub genes. The expression of these hub genes was further validated in an external dataset to obtain the critical mutually exclusive genes. Additionally, immune cell infiltration in psoriasis and leprosy was analyzed using single-sample gene set enrichment analysis (ssGSEA), and the correlation between critical mutually exclusive genes and immune cells was also examined. Finally, the expression pattern of critical mutually exclusive genes was evaluated in a single-cell transcriptome dataset. We identified 1098 DEGs in the leprosy dataset and 3839 DEGs in the psoriasis dataset. 48 candidate mutually exclusive genes were identified by taking the intersection. Enrichment analysis revealed that these genes were involved in cholesterol metabolism pathways. Through PPI network analysis, we identified APOE, CYP27A1, FADS1, and SOAT1 as hub genes. APOE, CYP27A1, and SOAT1 were subsequently validated as critical mutually exclusive genes on both internal and external datasets. Analysis of immune cell infiltration indicated higher abundance of 16 immune cell types in psoriasis and leprosy compared to normal controls. The abundance of 6 immune cell types in psoriasis and leprosy positively correlated with the expression levels of APOE and CYP27A1. Single-cell data analysis demonstrated that critical mutually exclusive genes were predominantly expressed in Schwann cells and fibroblasts. This study identified APOE, CYP27A1, and SOAT1 as critical mutually exclusive genes. Cholesterol metabolism pathway illustrated the possible mechanism of the inverse association of psoriasis and leprosy. The findings of this study provide a basis for identifying mechanisms and therapeutic targets for psoriasis.

CYBB-Mediated Ferroptosis Associated with Immunosuppression in Mycobacterium leprae-Infected Monocyte-Derived Macrophages.

Mycobacterium leprae-infected macrophages preferentially exhibit the regulatory M2 phenotype in vitro, which helps the immune escape unabated growth of M leprae in host cells. The mechanism that triggers macrophage polarization is still unknown. In this study, we performed single-cell RNA sequencing to determine the initial responses of human monocyte-derived macrophages against M leprae infection of 4 healthy individuals and found an increase in a major alternative-activated macrophage type that overexpressed NEAT1, CCL2, and CD163. Importantly, further functional analysis showed that ferroptosis was positively correlated with M2 polarization of macrophages, and in vitro experiments have shown that inhibition of ferroptosis promotes the survival of M leprae within macrophages. In addition, further joint analysis of our results with mutisequencing data from patients with leprosy and in vitro validation identified that CYBB was the pivotal molecule for ferroptosis that could promote the M2 polarization of M leprae-infected macrophages, resulting in the immune escape and unabated growth of pathogenic bacteria. Overall, our results suggest that M leprae facilitated its survival by inducing CYBB-mediated macrophage ferroptosis leading to its alternative activation and might reveal the potential for a new therapeutic strategy of leprosy.

Associação do poliformismo rs2228570 no gene VDR com o desfecho terapêutico em hanseníase / Association of rs2228570 polyformism in the VDR gene with therapeutic outcome in leprosy

A hanseníase é uma doença crônica que afeta principalmente os nervos periféricos e a pele, causada pelo Mycobacterium leprae, um parasita intracelular. O tratamento de primeira escolha na hanseníase é a poliquimioterapia (PQT) composta por rifampicina, dapsona e clofazimina. Estudos têm investigado a influência de fatores genéticos na suscetibilidade à hanseníase per se, porém não há investigações sobre a associação destes fatores com a resposta ao tratamento. Segundo os registros da OMS, o Brasil é o segundo país no mundo em número de casos retratamento da hanseníase. O gene do receptor de vitamina D (VDR) é um dos genes já associado com a doença. Além do papel importante no sistema imunológico, esse fator de transcrição, codificado por este gene, também atua no metabolismo de drogas. Assim, variações genéticas do tipo polimorfismos de nucleotídeo único (SNPs), no gene VDR podem afetar a resposta do organismo à doença e ao tratamento. Este estudo teve como objetivo avaliar a associação do polimorfismo rs2228570 no gene VDR com o desfecho terapêutico em casos de hanseníase multibacilar, através de estudo do tipo caso-controle. Foram analisados 315 prontuários de pacientes do estado de São Paulo, sendo 149 casos com necessidade de retratamento e 166 controles com sucesso terapêutico. A genotipagem do polimorfismo rs2228570 foi realizada por meio da técnica de discriminação alélica. A associação entre os genótipos e o desfecho terapêutico foi analisada por modelo de regressão logística multinomial, com ajuste dos dados pelas covariáveis sexo e etnia. Os resultados mostraram que o genótipo AA da variante rs2228570 no gene VDR está associado com o risco de retratamento na hanseníase multibacilar (Odds Ratio= 2.56; IC95: 1.13-5.82). Esse dado reafirma a importância da farmacogenética na terapêutica da hanseníase para a identificação de pacientes com maior risco de retratamento quando submetidos a poliquimioterapia convencional.

Leprosy is a chronic disease that mainly affects the peripheral nerves and skin, caused by Mycobacterium leprae, an intracellular parasite. The first-line treatment for leprosy is multidrug therapy (MDT) composed of rifampicin, dapsone, and clofazimine. Studies have investigated the influence of genetic factors on susceptibility to leprosy per se, but there have been no investigations into the association of these factors with treatment response. According to WHO records, Brazil is the second country in the world in the number of cases of leprosy retreatment. The vitamin D receptor (VDR) gene is one of the genes already associated with the disease. In addition to its important role in the immune system, this transcription factor, encoded by this gene, also plays a role in drug metabolism. Thus, genetic variations such as single nucleotide polymorphisms (SNPs) in the VDR gene can affect the body's response to the disease and treatment. This study aimed to evaluate the association of the rs2228570 polymorphism in the VDR gene with the therapeutic outcome in cases of multibacillary leprosy, through a case-control study. A total of 315 patient records from the state of São Paulo were analyzed, including 149 cases requiring retreatment and 166 controls with therapeutic success. Genotyping of the rs2228570 polymorphism was performed using the allele discrimination technique. The association between genotypes and therapeutic outcome was analyzed by multinomial logistic regression model, adjusting the data for covariates such as gender and ethnicity. The results showed that the AA genotype of the rs2228570 variant in the VDR gene is associated with the risk of retreatment in multibacillary leprosy (Odds Ratio= 2.56; IC95: 1.13-5.82). This data reaffirms the importance of pharmacogenetics in leprosy therapy for identifying patients at higher risk of retreatment when undergoing conventional multidrug therapy.

Detecção de mutações relacionadas a droga resistência no gene gyrB do Mycobacterium leprae / Detection of mutations related to drug resistance in the gyrB gene of Mycobacterium leprae

No Mycobacterium leprae (M. leprae) a resistência aos antimicrobianos dapsona (DDS), rifampicina (RIF) e ofloxacina (OFLO) se dá, primariamente, pela ocorrência de mutações em sequências conservadas dos genes folP1, rpoB e gyrA. Na rotina do Instituto Lauro de Souza Lima, muitos pacientes que apresentam clínica compatível com recidiva a qual poderia estar associada a resistência, apresentam perfil de suscetibilidade sensível a DDS, RIF e OFLO pelos mecanismos conhecidos. Existem vários outros mecanismos de resistência, bem como outros genes que podem ser pesquisados. Na rede de vigilância de resistência no Brasil, para fluorquinolonas, apenas as mutações em gyrA são pesquisadas na rotina, e, portanto, não temos dados sobre mutações em gyrB. No gene gyrB as mutações nos códons 214 (Val214Gly), 464 (Asp464Asn) e 503 (Thr503Ile) foram associadas com resistência à OFLO em M. leprae. O objetivo deste projeto é a detecção de mutações em gyrB por sequenciamento direto de DNA genômico de M. leprae. Para isso, foram utilizadas 52 amostras de DNA do banco de amostras do ILSL selecionadas entre julho de 2021 a dezembro de 2023, as quais já foram testadas por sequenciamento direto na rotina de investigação de resistência em hanseníase do ILSL para mutações já descritas. Foram utilizados dois pares de primers para amplificar e sequenciar as amostras pela metodologia de sequenciamento Sanger. As sequências foram analisadas utilizando-se o software Mega11. O Par 1, o qual permite avaliar polimorfismo no códon 214, enquanto que o Par 3, nos códons 464 e 503. As amostras eram em maioria (53,84%) do sexo masculino, 92,19% maiores de 20 anos com média da idade de 51 anos. Procedentes de vários estados brasileiros, com destaque para SP e MT. Cerca de 92,30% dos casos (48/52) eram multibacilares e 51,92% das amostras provenientes de pacientes com hanseníase virchowiana (MHV). Do total de casos, 55,70% foram associados a situações de falência terapêutica, seguida por casos novos, 19,23% e 11,54% de casos de recidiva da doença. A maioria (59,61%) fez PQT/MB, destes cerca de 74,19% trataram por 24 meses. O sequenciamento do gene gyrB pelo Par 1 foi eficiente em aproximadamente 98,07% dos isolados de M. leprae e pelo Par 3, 69,23%. Entretanto, nenhuma amostra foi polimórfica no gene gyrB e uma amostra apresentou polimorfismo não relacionado a droga resistência no códon 207 (Ile207Ile). Nossos resultados corroboram com a literatura, mostrando que mutações em gyrB é pouco frequente em M. leprae.

In Mycobacterium leprae (M. leprae), resistance to the antimicrobials dapsone (DDS), rifampicin (RIF), and ofloxacin (OFLO) primarily occurs due to mutations in conserved sequences of the folP1, rpoB, and gyrA genes. In the routine at the Lauro de Souza Lima Institute, many patients showing symptoms compatible with relapse, potentially associated with resistance, exhibit susceptibility profiles to DDS, RIF, and OFLO through known mechanisms. Numerous other resistance mechanisms and genes remain unexplored. In the Brazilian resistance surveillance network for fluoroquinolones, only gyrA mutations are routinely investigated, leaving a gap in data regarding gyrB mutations. Mutations at codons 214 (Val214Gly), 464 (Asp464Asn), and 503 (Thr503Ile) in the gyrB gene have been associated with OFLO resistance in M. leprae. The aim of this project is to detect gyrB mutations through direct genomic DNA sequencing of M. leprae. For this purpose, 52 DNA samples from the ILSL sample bank, selected between July 2021 and December 2023, were utilized. These samples had previously undergone routine direct sequencing at the ILSL for known mutations. Two primer pairs were employed to amplify and sequence the samples using Sanger sequencing methodology. Sequences were analyzed using Mega11 software. Primer 1, assessing polymorphism at codon 214, and Primer 3, targeting codons 464 and 503. The majority of samples (53.84%) were male, with 92.19% over 20 years old and an average age of 51 years. Originating from various Brazilian states, notably SP and MT, approximately 92.30% of cases (48/52) were multibacillary, and 51.92% of samples were from patients with virchowian leprosy (MHV). Among the cases, 55.70% were associated with therapeutic failure, followed by new cases (19.23%) and relapse cases (11.54%). The majority (59.61%) underwent PQT/MB treatment, with around 74.19% treated for 24 months. Sequencing of the gyrB gene using Primer 1 was effective in approximately 98.07% of M. leprae isolates, while Primer 3 showed efficiency in 69.23%. However, no sample exhibited polymorphism in the gyrB gene, and one sample presented non-drug resistance-related polymorphism at codon 207 (Ile207Ile). Our results align with the literature, demonstrating that gyrB mutations are infrequent in M. leprae.

Novel mutations found in Mycobacterium leprae DNA repair gene nth from central India.

INTRODUCTION: The importance of DNA repair enzymes in maintaining genomic integrity is highlighted by the hypothesis that DNA damage by reactive oxygen/nitrogen species produced inside the host cell is essential for the mutagenesis process. Endonuclease III (Nth), formamidopyrimide (Fpg) and endonuclease VIII (Nei) DNA glycosylases are essential components of the bacterial base excision repair process. Mycobacterium leprae lost both fpg/nei genes during the reductive evolution event and only has the nth (ML2301) gene. This study aims to characterize the mutations in the nth gene of M. leprae strains and explore its correlation with drug-resistance. METHOD: A total of 91 M. leprae positive DNA samples extracted from skin biopsy samples of newly diagnosed leprosy patients from NSCB Hospital Jabalpur were assessed for the nth gene as well as drug resistance-associated loci of the rpoB, gyrA and folP1 genes through PCR followed by Sanger sequencing. RESULTS: Of these 91 patients, a total of two insertion frameshift mutations, two synonymous and seven nonsynonymous mutations were found in nth in seven samples. Sixteen samples were found to be resistant to ofloxacin and one was found to be dapsone resistant as per the known DRDR mutations. No mutations were found in the rpoB region. Interestingly, none of the nth mutations were identified in the drug-resistant associated samples. CONCLUSION: The in-silico structural analysis of the non-synonymous mutations in the Nth predicted five of them were to be deleterious. Our results suggest that the mutations in the nth gene may be potential markers for phylogenetic and epidemiological studies.

Association of PTX3 gene polymorphisms and PTX3 plasma levels with leprosy susceptibility.

BACKGROUND: Pentraxin 3 (PTX3) is a soluble pattern recognition receptor that plays a crucial role in modulating the inflammatory response and activating the complement system. Additionally, plasma PTX3 has emerged as a potential biomarker for various infectious diseases. The aim of this study was to evaluate the association of PTX3 gene polymorphisms and PTX3 plasma levels with susceptibility to leprosy and clinical characteristics. METHODS: Patients with leprosy from a hyperendemic area in the Northeast Region of Brazil were included. Healthy household contacts and healthy blood donors from the same geographical area were recruited as a control group. The rs1840680 and rs2305619 polymorphisms of PTX3 were determined by real-time PCR. Plasma levels of PTX3 were determined by ELISA. RESULTS: A total of 512 individuals were included. Of these, 273 were patients diagnosed with leprosy; 53 were household contacts, and 186 were healthy blood donors. No association was observed between PTX3 polymorphisms and susceptibility to leprosy or development of leprosy reaction or physical disability. On the other hand, plasma levels of PTX3 were significantly higher in patients with leprosy when compared to household contacts (p = 0.003) or blood donors (p = 0.04). It was also observed that PTX3 levels drop significantly after multidrug therapy (p < 0.0001). CONCLUSIONS: Our results suggest that PTX3 may play an important role in the pathogenesis of leprosy and point to the potential use of this molecule as an infection marker.

Whole mitogenome sequencing uncovers a relation between mitochondrial heteroplasmy and leprosy severity.

BACKGROUND: In recent years, the mitochondria/immune system interaction has been proposed, so that variants of mitochondrial genome and levels of heteroplasmy might deregulate important metabolic processes in fighting infections, such as leprosy. METHODS: We sequenced the whole mitochondrial genome to investigate variants and heteroplasmy levels, considering patients with different clinical forms of leprosy and household contacts. After sequencing, a specific pipeline was used for preparation and bioinformatics analysis to select heteroplasmic variants. RESULTS: We found 116 variants in at least two of the subtypes of the case group (Borderline Tuberculoid, Borderline Lepromatous, Lepromatous), suggesting a possible clinical significance to these variants. Notably, 15 variants were exclusively found in these three clinical forms, of which five variants stand out for being missense (m.3791T > C in MT-ND1, m.5317C > A in MT-ND2, m.8545G > A in MT-ATP8, m.9044T > C in MT-ATP6 and m.15837T > C in MT-CYB). In addition, we found 26 variants shared only by leprosy poles, of which two are characterized as missense (m.4248T > C in MT-ND1 and m.8027G > A in MT-CO2). CONCLUSION: We found a significant number of variants and heteroplasmy levels in the leprosy patients from our cohort, as well as six genes that may influence leprosy susceptibility, suggesting for the first time that the mitogenome might be involved with the leprosy process, distinction of clinical forms and severity. Thus, future studies are needed to help understand the genetic consequences of these variants.

Mycobacterium leprae's Infective Capacity Is Associated with Activation of Genes Involved in PGL-I Biosynthesis in a Schwann Cells Infection Model.

Peripheral nerves and Schwann cells (SCs) are privileged and protected sites for initial colonization, survival, and spread of leprosy bacillus. Mycobacterium leprae strains that survive multidrug therapy show a metabolic inactivation that subsequently induces the recurrence of typical clinical manifestations of leprosy. Furthermore, the role of the cell wall phenolic glycolipid I (PGL-I) in the M. leprae internalization in SCs and the pathogenicity of M. leprae have been extensively known. This study assessed the infectivity in SCs of recurrent and non-recurrent M. leprae and their possible correlation with the genes involved in the PGL-I biosynthesis. The initial infectivity of non-recurrent strains in SCs was greater (27%) than a recurrent strain (6.5%). In addition, as the trials progressed, the infectivity of the recurrent and non-recurrent strains increased 2.5- and 2.0-fold, respectively; however, the maximum infectivity was displayed by non-recurrent strains at 12 days post-infection. On the other hand, qRT-PCR experiments showed that the transcription of key genes involved in PGL-I biosynthesis in non-recurrent strains was higher and faster (Day 3) than observed in the recurrent strain (Day 7). Thus, the results indicate that the capacity of PGL-I production is diminished in the recurrent strain, possibly affecting the infective capacity of these strains previously subjected to multidrug therapy. The present work opens the need to address more extensive and in-depth studies of the analysis of markers in the clinical isolates that indicate a possible future recurrence.

Interplay among differential exposure to Mycobacterium leprae and TLR4 polymorphism impacts the immune response in household contacts of leprosy patients.

Introduction: The aim of the present study was to investigate the association between the single nucleotide polymorphism (SNP) rs1927914 A/G in TLR4 gene and the immunological profile of household contacts (HHC) of leprosy patients. Leprosy classification is usually complex and requires the assessment of several clinical and laboratorial features. Methods: Herein, we have applied distinct models of descriptive analysis to explore qualitative/quantitative changes in chemokine and cytokine production in HHC further categorized according to operational classification [HHC(PB) and HHC(MB)] and according to TLR4SNP. Results and discussion: Our results showed that M. leprae stimuli induced an outstanding production of chemokines (CXCL8;CCL2; CXCL9; CXCL10) by HHC(PB), while increase levels of pro-inflammatory cytokines (IL-6; TNF; IFN-γ; IL-17) were observed for HHC(MB). Moreover, the analysis of chemokine and cytokine signatures demonstrated that A allele was associated with a prominent soluble mediator secretion (CXCL8; CXCL9; IL-6; TNF; IFN-γ). Data analysis according to TLR4 SNP genotypes further demonstrated that AA and AG were associated with a more prominent secretion of soluble mediators as compared to GG, supporting the clustering of AA and AG genotypes into dominant genetic model. CXCL8, IL-6, TNF and IL-17 displayed distinct profiles in HHC(PB) vs HHC(MB) or AA+AG vs GG genotype. In general, chemokine/cytokine networks analysis showed an overall profile of AA+GA-selective (CXCL9-CXCL10) and GG-selective (CXCL10-IL-6) axis regardless of the operational classification. However, mirrored inverted CCL2-IL-10 axis and a (IFN-γ-IL-2)-selective axis were identified in HHC(MB). CXCL8 presented outstanding performance to classify AA+AG from GG genotypes and HHC(PB) from HHC(MB). TNF and IL-17 presented elevated accuracy to classify AA+AG from GG genotypes and HHC(PB) (low levels) from HHC(MB) (high levels), respectively. Our results highlighted that both factors: i) differential exposure to M. leprae and ii) TLR4 rs1927914 genetic background impact the immune response of HHC. Our main results reinforce the relevance of integrated studies of immunological and genetic biomarkers that may have implications to improve the classification and monitoring of HHC in future studies.

Association of the rs2111234, rs3135499, rs8057341 polymorphisms in the NOD2 gene with leprosy: A case-control study in the Norte de Santander, Colombia population.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae. The development of leprosy involves several factors, including the causative agent, the individual host's immune response, environmental factors, and the genetic background of the host. Specifically, the host's innate immune response, encoded by genes, determines their susceptibility to developing leprosy post-infection. Polymorphic variants in the nucleotide-binding oligomerization domain 2 (NOD2) gene are associated with leprosy among populations in a variety of endemic areas around the world. Colombia, a country located in the tropical zone, has several leprosy-endemic regions, including Norte de Santander. The aim of this study was to analyze the rs7194886, rs2111234, rs3135499, and rs8057341 single nucleotide polymorphisms (SNPs) in the NOD2 gene using a case-control study to determine whether they confer greater or lesser susceptibility to the development of leprosy. METHODOLOGY: The TaqMan qPCR amplification system was used for SNPs detection. FINDINGS: An association between the A-rs8057341 SNP (p = 0,006286) and resistance to leprosy was found. However, the rs3135499 (p = 0,9063) and rs2111234 (p = 0.1492) were not found to be associated with leprosy susceptibility. In addition, the rs7194886 SNP was not found to be in Hardy-Weinberg equilibrium (HWE) in the study population. The GAG haplotype, consisting of SNPs rs2111234-G, rs3135499-A, and rs8057341G, acts as a susceptibility factor for the development of leprosy in women. SNPs rs3135499 and rs8057341 are functionally related to decreased NOD2 expression according to an in-silico analysis. CONCLUSIONS: The SNPs rs8057341-A was related with resistance to leprosy and the haplotype rs2111234-G, rs3135499-A and rs8057341-G SNPs was related with susceptibility in the Norte de Santander Colombia, studied population.

Allele-dependent interaction of LRRK2 and NOD2 in leprosy.

Leprosy, caused by Mycobacterium leprae, rarely affects children younger than 5 years. Here, we studied a multiplex leprosy family that included monozygotic twins aged 22 months suffering from paucibacillary leprosy. Whole genome sequencing identified three amino acid mutations previously associated with Crohn's disease and Parkinson's disease as candidate variants for early onset leprosy: LRRK2 N551K, R1398H and NOD2 R702W. In genome-edited macrophages, we demonstrated that cells expressing the LRRK2 mutations displayed reduced apoptosis activity following mycobacterial challenge independently of NOD2. However, employing co-immunoprecipitation and confocal microscopy we showed that LRRK2 and NOD2 proteins interacted in RAW cells and monocyte-derived macrophages, and that this interaction was substantially reduced for the NOD2 R702W mutation. Moreover, we observed a joint effect of LRRK2 and NOD2 variants on Bacillus Calmette-Guérin (BCG)-induced respiratory burst, NF-κB activation and cytokine/chemokine secretion with a strong impact for the genotypes found in the twins consistent with a role of the identified mutations in the development of early onset leprosy.

Evidence for Extensive Duplication and Subfunctionalization of FCRL6 in Armadillo (Dasypus novemcinctus).

The control of infections by the vertebrate adaptive immune system requires careful modulation to optimize defense and minimize harm to the host. The Fc receptor-like (FCRL) genes encode immunoregulatory molecules homologous to the receptors for the Fc portion of immunoglobulin (FCR). To date, nine different genes (FCRL1-6, FCRLA, FCRLB and FCRLS) have been identified in mammalian organisms. FCRL6 is located at a separate chromosomal position from the FCRL1-5 locus, has conserved synteny in mammals and is situated between the SLAMF8 and DUSP23 genes. Here, we show that this three gene block underwent repeated duplication in Dasypus novemcinctus (nine-banded armadillo) resulting in six FCRL6 copies, of which five appear functional. Among 21 mammalian genomes analyzed, this expansion was unique to D. novemcinctus. Ig-like domains that derive from the five clustered FCRL6 functional gene copies show high structural conservation and sequence identity. However, the presence of multiple non-synonymous amino acid changes that would diversify individual receptor function has led to the hypothesis that FCRL6 endured subfunctionalization during evolution in D. novemcinctus. Interestingly, D. novemcinctus is noteworthy for its natural resistance to the Mycobacterium leprae pathogen that causes leprosy. Because FCRL6 is chiefly expressed by cytotoxic T and NK cells, which are important in cellular defense responses against M. leprae, we speculate that FCRL6 subfunctionalization could be relevant for the adaptation of D. novemcinctus to leprosy. These findings highlight the species-specific diversification of FCRL family members and the genetic complexity underlying evolving multigene families critical for modulating adaptive immune protection.