3. Genetic variations in nucleotide-binding oligomerization domaincontaining protein 2 (NOD2)

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) is a family of intracellular PRR that is able to recognize bacterial molecules. Humans have 23 different NLRs that present two characteristic domains: NOD and leucine-rich repeats (LRRs). The first one is required for oligomerization, while the second one is required for ligand binding. There are different subfamilies of NLRs [48]. Some NLRs activate the protease caspase-1, so they act through inflammasomes leading to pyroptosis. The inflammasome-related NLRs are NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4, and NAIP. However, other components of NLR family (NOD1, NOD2, NLRP10, NLRX1, NLRC5, and CIITA) act through activation of NF-κB, mitogen-activated protein kinases (MAPKs), and interferon regulatory factors (IRFs) leading to innate immune response stimulation [4].

There are some PAMPs exclusively recognized by NLRs, such as muramyl dipeptide (MDP) that is a constituent of peptidoglycan cell wall present in Gram-positive and Gram-negative bacteria [49]. M. leprae, in turn, presents a distinct MDP structure, and some studies have evidenced the role of NOD2 in recognizing M. leprae through MDP [50]. NOD2-mediated recognition of MDP from M. leprae induces bacterial killing activating NF-κB signaling cascade, production of IL-32, differentiation of dendritic cells, and autophagy [51–53].

allele A of rs1927914 is associated with a higher expression of IL-17 and IL-1β, which is highly

The activity of TLRs must be under tight regulation to avoid an exacerbated response that would be harmful to the host. Toll-interacting protein (TOLLIP) is an adaptor protein which impairs the NF-κB and JNK signaling induced by TLR2 and TLR4, among other signaling pathways, playing a role in immune response regulation. TOLLIP is able to suppress IRAK-1 activity facilitating IL-1R/TLR-induced cell signaling during inflammation [44, 45]. After TLR2 stimulation, TOLLIP regulates the pro-inflammatory and anti-inflammatory balance by

In this way, changes in TOLLIP activity may be related to leprosy susceptibility. In a Mexican population, the possible association of TOLLIP polymorphisms with leprosy susceptibility was analyzed. The following four polymorphisms were investigated in lepromatous leprosy patients and control individuals: promoter 526 C > G (rs5743854), silent mutation rs3750920 (C/T P139P), rs5744015 (C/A A222S), and 3'UTR rs3750919 (G/A). However, no association was identified between the cited polymorphisms and lepromatous leprosy, but a trend of protective association to lepromatous leprosy was identified in homozygous CC genotype of

Six haplotype-tagging SNPs were also analyzed regarding association with leprosy in a population from Nepal. Among the tested SNPs, the intronic rs3793964 (C/T) exhibited association with leprosy being the TT genotype associated with leprosy susceptibility. Moreover, TT genotype and T allele were associated with increased expression of TOLLIP and IL-1Ra in the skin tissue. The study also demonstrates that TOLLIP induces IL-1Ra in monocytes that inhibit IL-1R activating helping M. leprae to evade from host immune response in early stages of

3. Genetic variations in nucleotide-binding oligomerization domain-

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) is a family of intracellular PRR that is able to recognize bacterial molecules. Humans have 23 different NLRs that present two characteristic domains: NOD and leucine-rich repeats (LRRs). The first one is required for oligomerization, while the second one is required for ligand binding. There are different subfamilies of NLRs [48]. Some NLRs activate the protease caspase-1, so they act through inflammasomes leading to pyroptosis. The inflammasome-related NLRs are NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4, and NAIP. However, other components of NLR family (NOD1, NOD2, NLRP10, NLRX1, NLRC5, and CIITA) act through activation of NF-κB, mitogen-activated protein kinases (MAPKs), and interferon regulatory factors (IRFs) leading to

produced in multibacillary leprosy patients [23].

150 Hansen's Disease - The Forgotten and Neglected Disease

inhibiting IL-6 and TNF and stimulating IL-10 [46].

rs3750920 [44].

leprosy development [47].

containing protein 2 (NOD2)

innate immune response stimulation [4].

2.4. Genetic variations in toll-interacting protein (TOLLIP)

Due to its role in immune response modulation, variations in NOD2 gene is involved with different inflammatory diseases, such as Crohn's disease [54], Blau's syndrome [55], arthritis [56], and others. In this sense, several studies have investigated the association of NOD2 genetic variants with leprosy susceptibility and leprosy reaction.

In a genome-wide association study from Zhang and coll. (2009) about susceptibility to leprosy in a Chinese population, variants in six genes of innate immune response were identified as associated with the disease (CCDC122, C13orf31, NOD2, TNFSF15, HLA-DR, and RIPK2). Specifically, in NOD2 gene, two variants were identified in association with susceptibility to the disease: rs9302752 (A/G) and rs7194886 (G/A), being the first one with a stronger association with multibacillary leprosy. The two other evaluated SNPs in NOD2, rs8057341 (A/G) and rs3135499 (A/C), exhibited no association with leprosy in this study [57]. After this genomewide association study, Grant and coll. (2012), in a family-based study, tried to validate in a Vietnamese population the genetic variants previously associated with leprosy in the Chinese study. They analyzed SNPs in HLA-DR-DQ, RIPK2, CCDC122-LACC1, and NOD2 in leprosy patients and control individuals. Four polymorphisms were evaluated in NOD2: rs9302752, rs7194886, rs8057341, and rs3135499. The G risk allele for the variant rs9302752 was associated with leprosy, as identified in Chinese population. According to this Vietnamese study, some variants identified by Zhang and coll. may not directly associated with leprosy risk, but they are in linkage disequilibrium with another variants, which are the causal ones. This possibility could explain the divergent results found in different studies [57, 58].

In a subsequent study, Marcinek and coll. (2013) genotyped the variant rs9302752 but now in an Indian population. However, this variant was not in Hardy-Weinberg equilibrium in this population. Some other variants in genes involved in signaling pathways mediated by NOD2 were shown to be associated with leprosy in this study. One of them is the haplotype formed by rs40457 G and rs42490 A in RIPK2 gene that together with NOD2 is responsible for activate NF-κB signaling. This haplotype gives an increased risk to leprosy in individuals, whereas the haplotype AA has a protective role. In addition, the minor A allele of rs1873613 polymorphism in LRRK2 is associated with paucibacillary leprosy progression [59].

Other conflicting results about genetic variants in NOD2 and their relation with leprosy emerged in a Brazilian study from Marques and coll. (2014) [60]. In this study, authors aimed to validate the results found in the previous Chinese study regarding SNPs in four genes: CCDC122-LACC1, NOD2, TNFSF15, and RIPK2 [57]. The A allele of variant rs8057341 in NOD2 was associated with leprosy resistance in Brazilian population. This result is also different from that identified in Vietnamese study, in which rs8057341 has no association with leprosy. Other variants from NOD2, besides rs8057341 A, were related to leprosy protection being under-transmitted to the affected offspring in Brazilian population: rs2111234 G and rs3135499 C [58, 60]. These conflicting results may be, at least partially, related to different genetic backgrounds among different populations.

Polymorphism rs751271 were already investigated in a Nepal population regarding its association with reactive states of leprosy showing no association with type 1 reversal reaction (RR) neither with erythema nodosum leprosum (ENL). In this study, 32 polymorphisms in NOD2 gene were evaluated. Four out of 32 polymorphisms were associated to leprosy susceptibility when the allele frequencies were compared between leprosy patients and healthy control: rs12448797, rs2287195, rs8044354, and rs1477176. When the genotype frequencies were compared, eight SNPs were associated with leprosy: rs2287185, rs8044354, rs8043770, rs13339578,

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Table 2. Association studies of genetic polymorphisms in NOD2 and leprosy.

The previous study conducted by Zhang and coll. was realized in a Han Chinese population, which is the main ethnic group from China [57, 61]. A second study was realized in a Chinese population evaluating the genetic association of NOD2 (C13orf31 and CCDC122) with leprosy but now in a population from Yi ethnic group that represents a minority group. The following polymorphisms in NOD2 were investigated: rs9302752, rs7194886, rs8057341, and rs3135499. Only the SNP rs3135499 was differentially distributed between leprosy patients and healthy control individuals [62]. This result is different from that found for rs9302752 by Zhang and coll., Marcinek and coll., and Grant and coll. and rs7194886 and rs8057431 by Zhang and coll. [57–59].

Even with some discrepant results, the cited studies agree in associate NOD2 with leprosy, however, with different specific variants. These differences may be due to the influence of different ethnicities or linkage disequilibrium between different SNPs.

In addition to association with susceptibility to leprosy, variation in NOD2 may be related to susceptibility to development of leprosy reactions. Leprosy reactions are acute inflammatory reactions that may occur before leprosy diagnosis, during the treatment or after the treatment. Some factors are able to initiate these reactions, like intercurrent infections, pregnancy, and physical and emotional stress, among others [63]. Leprosy reactions are classified into two main types: type 1—reversal reaction (RR) and type 2—erythema nodosum leprosum (ENL). RR occurs mainly in earlier stages of treatment but can also occur after the treatment, in borderline clinical forms. It is characterized by an exacerbated Th1 response. The patient with RR presents an exacerbation of preexisting lesions, which became edematous and erythematous and may develop ulceration. RR is the leading cause of nerve damage in leprosy and consequent physical disability [63–65]. ENL, in turn, mainly occurs in lepromatous and borderline lepromatous leprosy and may happen during treatment but is more frequent after the treatment. Patients present subcutaneous nodules that are painful and erythematous and also may be ulcerative. In this kind of reaction, immune complexes are related to their initiation, but cell-mediated immunity also plays a role in ENL. There is an increase in CD4+ T cells, TNFα, and IFN-γ [63, 66, 67]. Several studies have already evidenced the possible functional mechanisms by which NOD2 plays a role during development of leprosy reactions by proinflammatory activities. The stimulation of Th1 and Th2 is one of these mechanisms [68, 69], as well as negative regulation of TLR2-mediated Th1 response [33, 70, 71].

Some of the previously mentioned polymorphisms in NOD2 (rs7194886, rs9302752, and rs8057341), besides the additional rs751271 (G/T) and rs2066843 (C/T), were investigated for their association with leprosy inflammatory reactions in a Brazilian population. The variant rs751271 is associated with leprosy, being the genotype TT related to faster reaction development, whereas the genotype GT and G allele carriers are protection factors against leprosy reactions. The authors also suggest that rs751271-GT genotype produces lower levels of IL-6 in patients without reaction, so the hypothesis is that individuals with this genotype present a better balance in cytokine production which could be related to protection against leprosy reactions [72].

Polymorphism rs751271 were already investigated in a Nepal population regarding its association with reactive states of leprosy showing no association with type 1 reversal reaction (RR) neither with erythema nodosum leprosum (ENL). In this study, 32 polymorphisms in NOD2 gene were evaluated. Four out of 32 polymorphisms were associated to leprosy susceptibility when the allele frequencies were compared between leprosy patients and healthy control: rs12448797, rs2287195, rs8044354, and rs1477176. When the genotype frequencies were compared, eight SNPs were associated with leprosy: rs2287185, rs8044354, rs8043770, rs13339578,

rs3135499 C [58, 60]. These conflicting results may be, at least partially, related to different

The previous study conducted by Zhang and coll. was realized in a Han Chinese population, which is the main ethnic group from China [57, 61]. A second study was realized in a Chinese population evaluating the genetic association of NOD2 (C13orf31 and CCDC122) with leprosy but now in a population from Yi ethnic group that represents a minority group. The following polymorphisms in NOD2 were investigated: rs9302752, rs7194886, rs8057341, and rs3135499. Only the SNP rs3135499 was differentially distributed between leprosy patients and healthy control individuals [62]. This result is different from that found for rs9302752 by Zhang and coll., Marcinek and coll., and Grant and coll. and rs7194886 and rs8057431 by Zhang and coll.

Even with some discrepant results, the cited studies agree in associate NOD2 with leprosy, however, with different specific variants. These differences may be due to the influence of

In addition to association with susceptibility to leprosy, variation in NOD2 may be related to susceptibility to development of leprosy reactions. Leprosy reactions are acute inflammatory reactions that may occur before leprosy diagnosis, during the treatment or after the treatment. Some factors are able to initiate these reactions, like intercurrent infections, pregnancy, and physical and emotional stress, among others [63]. Leprosy reactions are classified into two main types: type 1—reversal reaction (RR) and type 2—erythema nodosum leprosum (ENL). RR occurs mainly in earlier stages of treatment but can also occur after the treatment, in borderline clinical forms. It is characterized by an exacerbated Th1 response. The patient with RR presents an exacerbation of preexisting lesions, which became edematous and erythematous and may develop ulceration. RR is the leading cause of nerve damage in leprosy and consequent physical disability [63–65]. ENL, in turn, mainly occurs in lepromatous and borderline lepromatous leprosy and may happen during treatment but is more frequent after the treatment. Patients present subcutaneous nodules that are painful and erythematous and also may be ulcerative. In this kind of reaction, immune complexes are related to their initiation, but cell-mediated immunity also plays a role in ENL. There is an increase in CD4+ T cells, TNFα, and IFN-γ [63, 66, 67]. Several studies have already evidenced the possible functional mechanisms by which NOD2 plays a role during development of leprosy reactions by proinflammatory activities. The stimulation of Th1 and Th2 is one of these mechanisms [68, 69], as

Some of the previously mentioned polymorphisms in NOD2 (rs7194886, rs9302752, and rs8057341), besides the additional rs751271 (G/T) and rs2066843 (C/T), were investigated for their association with leprosy inflammatory reactions in a Brazilian population. The variant rs751271 is associated with leprosy, being the genotype TT related to faster reaction development, whereas the genotype GT and G allele carriers are protection factors against leprosy reactions. The authors also suggest that rs751271-GT genotype produces lower levels of IL-6 in patients without reaction, so the hypothesis is that individuals with this genotype present a better balance in cytokine production which could be related to protection against leprosy

different ethnicities or linkage disequilibrium between different SNPs.

well as negative regulation of TLR2-mediated Th1 response [33, 70, 71].

genetic backgrounds among different populations.

152 Hansen's Disease - The Forgotten and Neglected Disease

[57–59].

reactions [72].


Table 2. Association studies of genetic polymorphisms in NOD2 and leprosy.

rs4785225, rs751271, rs12448797, and rs1477176. The variants rs12448797 and rs1477176 are located in genes adjacent to NOD2, SL1C1, and CYLD, which are regions in linkage disequilibrium with NOD2 that may influence the expression of SL1C1 or CYLD and NOD2. Regarding to RR, five SNPs showed association of their allele frequencies (rs2287195, rs8044354, rs8043770, rs7194886, and rs1861759), and seven SNPs exhibited association of their genetic frequencies in a dominant model (rs2287195, rs8044354, rs8043770, rs7194886, rs1861759, rs4785225, and rs751271). Four SNPs were associated with ENL in an allelic level (rs8044354, rs17312836, rs1861758 and rs1861759), while seven SNPs at the genotypic level (rs2287195, rs8044354, rs7194886, rs6500328, rs17312836, rs1861759, and rs18611758) [73].

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The association of polymorphisms in NOD2 gene with leprosy reactions highlights the potential role of this gene in susceptibility and development of RR and ENL. The genetic association studies between NOD2 and leprosy are summarized in Table 2.
