Influence of Genetic Ancestry on INDEL Markers of NFKβ1, CASP8, PAR1, IL4 and CYP19A1 Genes in Leprosy Patients

Background Leprosy is an insidious infectious disease caused by the obligate intracellular bacteria Mycobacterium leprae, and host genetic factors can modulate the immune response and generate distinct categories of leprosy susceptibility that are also influenced by genetic ancestry. Methodology/Principal Findings We investigated the possible effects of CYP19A1 [rs11575899], NFKβ1 [rs28362491], IL1α [rs3783553], CASP8 [rs3834129], UGT1A1 [rs8175347], PAR1 [rs11267092], CYP2E1 [INDEL 96pb] and IL4 [rs79071878] genes in a group of 141 leprosy patients and 180 healthy individuals. The INDELs were typed by PCR Multiplex in ABI PRISM 3130 and analyzed with GeneMapper ID v3.2. The NFKβ1, CASP8, PAR1 and IL4 INDELs were associated with leprosy susceptibility, while NFKβ1, CASP8, PAR1 and CYP19A1 were associated with the MB (Multibacilary) clinical form of leprosy. Conclusions/Significance NFKβ1 [rs28362491], CASP8 [rs3834129], PAR1 [rs11267092] and IL4 [rs79071878] genes are potential markers for susceptibility to leprosy development, while the INDELs in NFKβ1, CASP8, PAR1 and CYP19A1 (rs11575899) are potential markers for the severe clinical form MB. Moreover, all of these markers are influenced by genetic ancestry, and European contribution increases the risk to leprosy development, in other hand an increase in African contribution generates protection against leprosy.


Introduction
Leprosy is an insidious infectious disease caused by the obligate intracellular bacteria Mycobacterium leprae that affects the skin and peripheral nerves, causing a chronic granulomatous infection [1]. Leprosy patients may be classified in two major groups, based on clinical manifestations using a simple system introduced by the WHO (World Health Organization) in 1982. Paucibacillary (PB) is the primary characteristic of Tuberculoid (TT) leprosy and is characterized by a few lesions and scarce bacilli, and Multibacillary (MB) is the primary characteristic of anergic Lepromatous (LL) leprosy. From an epidemiological perspective, the situation in Brazil is critical because, along with India and Indonesia, it has the highest rate of new cases detected worldwide [2,3,4].
In addition to the system introduced by WHO in 1982, the use of histological and immunological criteria as described by Ridley-Jopling further improves definition of Borderline cases. According to this classification, TT (tuberculoid-tuberculoid) patients, who have the PB type, exhibit a strong cellular immune response (CIR) mediated by Th1, and a negative skin smear test. In contrast, LL (lepromatous-lepromatous) patients have a weak or absent CIR and a highly positive skin smear associated to an humoral immune response. In the middle of this spectrum are a large number of borderline patients, which together with LL comprise the MB pole, with symptoms varying from weak to strong CIR and negative to positive skin smears [5,6].
The regulation of the host immune response and manifestation of disease clinical between types PB (better) and MB (severe) involves cytokine and others mediators produced by various subtypes of T cells. In PB, an inflammatory immune response is mediated by Th1 cells that express pro-inflammatory interleukins that stimulate macrophages and phagocytosis mechanisms to inhibit bacillary growth and kill mycobacteria [2,[7][8][9]. On the other hand, MB patients have an intense Th2 immune response with production of anti-inflammatory cytokines in addition to the specific anti-PGL-1 (phenolic glycolipid 1) antibody. This mechanism does not block bacillary growth and contributes to the host's inability to resist the development of severe disease [2,8,[9][10][11].
Recent studies have investigated genetic markers, usually innate immune response genes, as possible susceptibility factors for leprosy because the SNPs in these genes can modulated the host immune response and consequently lower host resistance to bacillus growth [6,12,13]. However, few studies have investigated INDEL polymorphisms (insertion-deletion) in immune response genes in leprosy. Moreover, such polymorphisms present interesting features as genetic markers because i) INDELs are spread throughout the human genome, ii) INDELs derive from a single event (they do not present homoplasy), iii) small INDELs can be analyzed using short amplicons, which improves amplification of degraded DNA and facilitates multiplexing reaction, iv) INDELs can create abrupt changes in the normal function of the gene and v) INDELs can be easily genotyped using a simple dye-labeling electrophoretic approach [14]. The current study select eight INDEL in seven genes (CYP19A1, NFKβ1, IL1α, CASP8, UGT1A1, PAR1, CYP2E1, and IL4), which have relation with the immune response modulation in leprosy patients, beside literature that demonstrate these molecular markers like functional polymorphisms that alter transcriptional activity of the gene, and consequently the immunological phenotype against the bacilli. Additionally these INDELs can be able to contribution to construction a possible panel of susceptibility markers.
However, from the genetic point of view, Brazil is recognized as having one of the most heterogeneous populations in the world, with important genetic information being contributed by three main continental groups, Europeans, Africans and Amerindian, resulting in a genetically very diverse modern Brazilian population [15]. Therefore, analysis of genetic markers in complex diseases may result in spurious results due to population substructure [16], and it is important to perform the genomic ancestry control, especially in populations with a high degree of interethnic admixture [14].

Ethics statement
The project was approved by the Pará Federal University ethics committee (N°197/07).

Samples
We investigated 141 leprosy patients who attended the Dr Marcello Candia Reference Unit in Sanitary Dermatology of the State of Pará (UREMC), in Marituba, Pará, Brazil between January 2008 and December 2009. All patients were informed about the study before they signed informed consent forms. Since 2002, UREMC registered between 308 and 472 leprosy patients (mean: 408 cases per year). Of the 765 leprosy cases registered in 2008 and 2009 alone, 141 (18.43%) were randomly selected for this study. These patients were divided according to Ridley-Jopling classification [5] into Paucibacillary (TT: PB 31) and Multibacillary (BT, BB, BL and LL: MB 110) groups. A total of 180 healthy individuals who were unrelated, without leprosy or other chronic diseases and from the same geographic area as each other were chosen for the control group. Leprosy patient's descriptions were made previously [6]. These subjects were asked to participate in the study after being informed about the study objectives and signing informed consent forms.
Multiplex Typing. DNA samples were typed for the 7 biallelic INDELs and 1 VNTR of 70 bp. Each multiplex PCR was performed in a final volume of 10 μL containing 5 μL of QIAGEN Multiplex PCR Kit, 1 μL of Q-solution, 1 μL of primer mix (Forward + Reverse primers at a concentration of 2 μM each), 1 μL of DNA (10 ng) and 2 μL of water. The fluorescent molecules 6FAM and HEX were inserted at the 5' position of each primer (forward or reverse). All PCR reactions were performed on the Thermocycle Veriti-96 Well Thermal Cycler (Life Technologies, CA, USA). Before capillary electrophoresis, 1.0 μL PCR product was added to 8.5 μL deionized formamide HI-DI (Life Technologies, CA, USA) and 0.5 μL GeneScan 500 LIZ size standard (Life Technologies, CA, USA). DNA fragments were separated using an ABI PRISM 3130 Genetic Analyzer (Life Technologies, CA, USA) and analyzed with GeneMapper ID v3.2 software (Life Technologies, CA, USA). This method is similar like described in a paper of INDEL markers of our group [14].

Ancestry Informative Marker (AIM)
Individual interethnic admixture was estimated using a panel of 48 ancestry informative markers (AIMs) as previously described [6,14].

Statistical analysis
The allelic frequencies between healthy individuals and leprosy patients and between PB and MB patients were estimated by gene counting. Deviation from the Hardy-Weinberg equilibrium was assessed using chi-squared tests, using the Arlequin v3.5 software [18], and p-value of HWE was corrected by Bonferroni methods.
Differences between leprosy patients and healthy individuals and between PB and MB patients with respect to age, gender and genetic ancestry were estimated using Student's t-Test, Fisher's exact test and Mann-Whitney tests, respectively. The association of markers between groups was analyzed by logistic regression tests, all the test were corrected by FDR (False Discovery Rate) method, and all tests were performed using the statistical package under R calculation. A two-tailed p-value < 0.05 was considered statistically significant.
The individual contributions of European, African and Amerindian genetic ancestry were estimated using the STRUCTURE 2.3.3 program assuming three parental populations (European, African and Amerindian), a burn-in period of 200,000, and 200,000 Markov Chain Monte Carlo repetitions after burn-in [16]. The differences in allelic frequencies between leprosy cases and the healthy individuals for markers analyzed following an adjustment for population stratification was performed using the STRAT software program with 10,000 simulations [16].

Results
The data of clinical and demographic distribution of leprosy patients and healthy individuals is shown in Table Table 2. For the polymorphism in IL4 (VNTR of 70 bp), only two alleles were identified in the sample. One allele had two repeats of 70 bp (allele A1) and the other had three repeats of 70 bp (allele A2), suggesting theses alleles are biallelic markers. All the polymorphisms analyzed were according to the Hardy Weinberg equilibrium, therefore the association analysis were performed with regression logistic test and differences in allelic frequencies were corrected by frequencies of ancestry markers informative.
When the INDELs were analyzed by logistic regression, the genes NFKβ1 and PAR1 showed statistically significant differences associated with the presence of the DEL allele (p = 0.016 and p = 0.022, respectively) and both were associated like protection factors to not developing the disease (OR[IC95%] = 0.50[0.27-0.88] and OR[IC95%] = 0.35[0.14-0.86], respectively), for these genes was found a dominance effect DEL allele, that increase your protection capacity in general population. The CASP8 showed significant differences associated with the presence of the DEL/DEL homozygous genotype and was associated with a risk factor for leprosy development (p = 0.017; OR[IC95%] = 2.33 [1.16-4.69]) ( Table 3). The analysis of allele frequency differences was then corrected for the influence of genetic ancestry on population structure, and the results showed that the DEL allele of PAR1 gene and the allele A 1 of IL4 is more frequent in healthy individuals (p = 0.018 and p = 0.019, respectively) ( Table 3), these results shown the importance of statistical correction in admixture population, in order to exhibit differences covert by structure population. Table 4 summarizes the clinical and demographic characteristics of leprosy patients grouped according to clinical manifestation in PB (Paucibacillary) and MB (Multibacillary) groups, and the only significant difference was observed for age (p = 0.003), with a higher mean age in MB patients (45.7±22 versus 34.9±15). When the INDELs were analyzed by logistic regression, NFKβ1 showed significant differences like risk factor associated with the presence of the allele DEL in MB patients (p = 0.024; OR[IC95%] = 2.64[1.13-6.19]), of contradictory way the dominance effect of DEL allele seem protect against the development of leprosy, but when the disease is established your effect seem inefficient to combat to bacilli. PAR1 showed significant differences associated with the presence of homozygous DEL/DEL genotype in PB patients (p = 0.031; OR[IC95%] = 0.41[0.17-0.96]) ( Table 5). The analysis of allele frequency differences were corrected for population structure and showed that the DEL allele of CASP8 is more frequent in PB patients (p = 0.003), while the DEL allele of CYP19A1 is more frequent in MB patients (p = 0.007) ( Table 5).  These results are better understood on frequencies distribution, according with range of ancestry contribution (S1 Table). For African ancestry 99.4% of health individual is closed between 0% and 50% of African contribution (range that have p<0.05 on Fig 1), moreover the contribution range of 10% to 30% is closed 81.7% of health individual, in this range the Fig 1 have more decline of OR value, that showed the higher protection effect of African ancestry. To European ancestry, 61.7% of leprosy patients is closed between 40% to 80% of European contribution, while 87.2% of health individuals is closed between 0% to 50% of European contribution. Additionally, for the contribution range between 60% to 80% we observed 17% of all patients, while no healthy individual was observed this range, these data show that leprosy patients have higher European contribution compared with healthy individuals. Take together

Discussion
NF-κB belongs to family of protein transcription factors that modulate many inflammatory processes. In the resting state, IκBα (inhibitor of NF-kβ activity) sequesters NF-κB in the cytoplasm and prevents its activity, but in response to specific stimuli, IkBα is ubiquitinated and degraded allowing NF-kB to migrate to the nucleus and stimulate the transcription of proinflammatory genes [19,20]. The allele DEL (rs28362491) has been shown to be associated with a decrease of transcriptional activity of variety genes of immune response [21] and with auto immune disease such as Systemic Sclerosis [22] and lupus erythematosus [23]. The role of NF-kβ in leprosy is not clear, and studies linked to expression of NF-kβ have suggested that lower expression is common in leprosy patients [24,25]. Our results suggest that the DEL carries genotype induces protection against leprosy (Table 3), although a comparison of PB and MB patients also suggests that DEL behaves like a risk factor for the development of the severe clinical form of MB (Table 5). Because the transcription of NF-kβ is mediated by specific stimuli, such as the presence of M. leprae [24], it is conceivable that the presence of DEL confers risk to MB leprosy.
PAR1 is a receptor of the PAR family of proteins that belong to a unique group of G protein -coupled receptors. In particular, PAR1 protein is present in a variety of cells like platelets, endothelia, epithelial, neurons, fibroblasts, smooth muscle, leukocytes and tumor lines [26]. This receptor has been shown to be involved in many natural physiological processes, that involve inflammation like the systems cardiovascular, respiratory and central nervous and in embryogenesis, cancer and inflammation [27]. PAR1 suppresses T helper type 1 (Th1) and T helper type 17 (Th17) cells and the secretion of IL-12 and IL-23, thereby resulting in the inhibition of pro-inflammatory responses [28]. The allele of insertion (INS) of INDEL studied (rs11267092) has been shown to increase gene transcription [29] and therefore, it is a risk allele for leprosy. Our results suggest that the presence of DEL induces protection against leprosy (Table 3), and the DEL/DEL genotype confers protection against the development of clinical forms of MB (Table 5), thus this genotype of PAR1 gene can suppresses cellular infiltration and increase both Th1 and Th17 responses to infection. Moreover, analyses of macrophages revealed that secretion of IL-12 and IL-13, two cytokine that play role key on cellular immunity Th1 and Th17, can be suppressed by PAR1 activation. Furthermore, PAR1 can suppress interferon regulatory factor 5 (IRF5), that play role key like transcription factor for IL-12 and IL-23, which modulates the sub sets of cellular immunity. Thereby the suppression of IRF5 and IL-12/23 secretion by PAR1 gene, can provides a novel mechanism by which the host suppresses the Th1 and Th17response to infection, and dysregulation of this process can likely an important factor in the susceptibility of some individuals to leprosy [28].
Macrophages with a high load of M. leprae have been shown to undergo apoptosis, and this mechanism is under the control of cytokines [30]. In leprosy patients, the immune system is overburdened with bacilli, and most likely the continuous activation of T cells by circulating M. leprae antigens leads to apoptosis and to a reduction of peripheral lymphocytes and other immune effector cells in these patients with the regulation of apoptosis involved in the stimulation and activation of caspase-8 [31]. The allele DEL (rs3834129) cause a decrease in CASP8 transcription and a reduction in apoptosis [32], thereby improving the bacillary load. Our results suggested that the DEL/DEL genotype (Table 3) and the high frequency of DEL allele (Table 5) can raise the bacillary load and thus confers a risk to leprosy development.
Interleukin-4 (IL-4) is a key cytokine secreted by Th2 lymphocytes, eosinophils and mast cells that induces the activation and differentiation of B cells and the development of the Th2 subset of lymphocytes, which is ineffective in combating leprosy [33]. Our analysis of the VNTR on intron 3 of the IL4 gene (rs79071878) revealed two common alleles with two (A 1 ) and three (A 2 ) tandem repeats. Of these, A 2 allele is known to be a high producer of IL-4 [34].
Our results indicate that allele A 2 is more frequent in leprosy patients compared to healthy individuals, consistent with the fact that higher levels of IL4 would be ineffective in controlling the growth of bacilli ( Table 3).
The conversion of androgens to estrogens, catalyzed by aromatase encoded by the CYP19A1 gene, is the primary pathway of estrogen production in humans [35]. The levels of these hormones are important in leprosy patients and it has been demonstrated that androgen levels are significantly lower in leprosy patients compared to healthy control subjects [36]. Moreover, there is an inverse correlation between plasma androgen levels and secretion of inflammatory cytokines, suggesting that high plasma androgen levels can be less effective in inhibiting bacillus growth [37]. The DEL allele (rs11575899) has previously been reported to have a negative effect on aromatase activity [38], and our results show that the DEL allele is more frequent in MB patients (Table 5). We hypothesize that the DEL allele can decrease aromatase activity and increase androgen levels, resulting in an overall reduction in effective combat of bacillary growth and development of the severe clinical form MB. It is unclear whether leprosy originated in Asia or Africa. However, leprosy is believed to have been introduced into Europe from India, and the incidence was high in Europe during the Middle Ages until approximately 1870 when the number of cases dramatically reduced because of socioeconomic development [39,40,41]. It is believed that leprosy was introduced in Brazil primarily by the Spanish and Portuguese [41]. Estimates indicate that before the arrival of colonizers, approximately 2.5 million natives lived in Brazil, and during the European immigration in the first three centuries, approximately 500,000 individuals came from Portugal and approximately 3.5 million Africans were brought into Brazil through slave trade [14]. Therefore, there is evidence of a so-called directed admixture process involving predominantly European, Native American and African people [42][43][44][45].
Our data indicates that the contribution of different ethnic groups to the composition of the current Brazilian population can generate different rates of risk for leprosy development according to the level of inter-ethnic composition of the individuals involved. Our analysis suggests that an increase in European contribution increases the risk of leprosy development, while an increase in African contribution decreases the risk for leprosy development and the Amerindian contribution does not result in any statistically significant differences (Fig 1).
The introduction of leprosy in Brazil primarily can it be accredited to the slave trade, but no only for this reason. Slaves were firstly there from Africa, and in succeeding years the number these slaves were increased, but was not common between they the clinical manifestation of leprosy, because these slaves were from region of the Africa where leprosy was comparatively rare. Moreover isn't doubt that the Portuguese and, to a less degree, Dutch, French and Spaniards were responsible by introduction of leprosy in Brazil, on period of country colonization. Additionally, data showed that as early, as 1419, the disease was common in Portuguese and epidemiologically in this time the leprosy was very prevalent in Europe, and particularly in Portugal [41]. Therefore, our data of risk of leprosy according the different ethnic groups compositions is consistent with the higher numbers of settlers Portuguese that came to Brazilian that probably increases the frequencies of alleles of susceptibility on Brazilian population [14,[42][43][44][45]. In other hand, the African contribution may have increase the frequencies of allele that confer protection against to leprosy.
Comparative analyses of the four M. leprae genomes (India, Thailand, Brazil and US) have revealed little clonal differences. Thus, the patterns of global human migration routes, during the past 100,000 years, corroborate and suggest that leprosy probably originated in Africa [46]. African-descendants in admixture populations can be less susceptible to the leprosy bacilli, probably because of genetic polymorphisms accumulated during these times, in gene that can modulate the immune response on infection combat. Furthermore African humans are the more genetically diverse population in the world consequently, by selection bias, genetic polymorphisms accumulated that confer protection against disease, can be present in this population and your descendants.
Of point view epidemiological, the situation of African and Americas region is critical, and is associated the socioeconomic challenges related to the disease, but genetics components also are important to disease knowledge [4]. Thus understanding of like genetic ancestry, in admixture population, can to influence genetic susceptibility is essential to avoid spurious results. In conclusion, our study shows that the NFKβ1 [rs28362491], CASP8 [rs3834129], PAR1 [rs11267092] and IL4 [rs79071878] genes are possible markers for the susceptibility to development of leprosy and the severe clinical form MB. Moreover, after correcting for population structure within an admixture population, the results show that different levels of ethnic group composition can generate different OR rates for leprosy susceptibility.
Supporting Information S1 Table. Distribution of percent range of African and European genetic ancestry between leprosy patients and healthy individuals. (DOCX)