INTRODUCTION 1.1 INTRODUCTION

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1 1.1 Mycobacterium leprae is the causative agent of human leprosy. The disease, an ancient affliction of humankind, has persisted into contemporary times despite the fact that it is not highly transmissible and the chemotherapy has been available for a long period (Misch et al. 2010; Nordeen 1985). Since the introduction of multidrug therapy (MDT) in 1982 by the World Health Organisation, the leprosy burden has been dramatically reduced, however over the past 5 years more than 250,000 new cases have continued to be detected and reported each year (Rodrigues et al. 2010). Although the reduction of afflicted cases over decades partly reflects improved leprosy control but the continuing detection of new cases in endemic countries (Lockwood 2002) probably indicates that the global use of MDT had limited impact on the transmission cycle of M. leprae (Alcais et al. 2005). Leprosy is a spectral disease in which pathology and immunology are inextricably related. The study of the disease provides a unique critical model to explore the immunoregulatory mechanisms in humans. Patients with tuberculoid leprosy have a few localized lesions containing rare organisms. A strong cell-mediated immune response is directed against Mycobacterium leprae antigens that ultimately kill and clear the bacilli, although often with concomitant injury to nerves. In contrast, lepromatous leprosy patients have numerous skin lesions containing extraordinarily high numbers of acid-fast bacilli with specific immunological unresponsiveness to antigens of M. leprae in vivo and in vitro (Danielssen et al. 1847). Phenotyping of lymphocytes in leprosy lesions (Gansert et al. 2003; Modlin and Rea 1988; Salgame et al. 1991; Sieling 2000; Sieling et al. 1999; Sieling et al. 2005; Yamamura et al. 1991), the selective accumulation of lymphocytes, their antigen specificity, and immunological functions of different lymphocyte subsets have shown striking dichotomy with Th1 type response in tuberculoid patients and Th2 type profile in lepromatous cases. The circulating lymphocyte responses to M. leprae antigens, however, do not show such distinct polarization across the spectrum, reflecting differential activation status of patients across the spectrum to chronic M. leprae infection (Howe et al. 1995; Misra et al. 1995). The gene expression profiles of tuberculoid and lepromatous cutaneous lesions have been found to be consistent with the bias in type 1 and type 2 cytokine production between the leprosy subtypes (Bleharski et al. 2003). The fundamental question regarding the specific unresponsiveness of lepromatous patients to M. leprae antigens and the strategies employed Page 1

2 by the pathogen to subvert immune response in immunocompromised leprosy patients still remains unanswered (Figure 1.1). Figure 1.1: Clinical and immunological classification of the leprosy disease spectrum Clinical manifestations of leprosy are classified according to the Ridley-Jopling (TT, BT, BB, BL, LL) and World Health Organization (PB and MB) schemes. The host immune response (Th1 versus Th2) correlating with each leprosy subtype and leprosy reactions: reversal and erythema nodosum leprosum (ENL), are indicated. (modified from (Britton and Lockwood 2004)). Investigation of mechanisms underlying infection, pathogenesis, etiology and clinical sequel has been limited by the fact that M. leprae infects only humans and cannot be cultured in vitro. However, the complete sequence of the genome of M. leprae was published at the beginning of the 21st century (Cole et al. 2001). Comparative genomics of M. tuberculosis and M. leprae have shown strong gene decay in M. leprae, resulting in the loss of entire metabolic pathways present in M. tuberculosis. At 3.27 Mb, the genome of M. leprae is substantially smaller than that of M. tuberculosis and a mere 49.5% is occupied by the 1,605 protein-coding genes (Cole et al. 2001). This probably renders the leprosy bacillus dependent Page 2

3 on host metabolic products and might explain its long generation time and inability to grow in culture. M. leprae has probably lost 2,000 genes during its evolution and the minimal gene set required by a pathogenic mycobacterium appears to have been defined naturally. The comparative genomic studies are being intensely pursued to identify novel specific drug targets for effective therapeutic intervention and new subunit vaccine candidates defined with higher efficacy. The molecular epidemiological studies using the M. leprae genome information have shown that different M. leprae isolates exhibit very limited genetic diversity (Monot et al. 2005; Monot et al. 2009). Though comparative genomics has provided better insights, the inability to culture M. leprae in vitro and the lack of a suitable animal model have hampered leprosy research such that the direct experimental evidences to understand host-m. leprae interaction and mechanisms behind M. leprae survival within macrophages, are still limited. Forward genetics has been the main method used to identify susceptibility variants in genes, and consequently the immunological pathways involved in the human response to M. leprae (Alcais et al. 2005), due to an extensive gene decay and unavailability of animal model of leprosy disease. Forward genetics includes several study designs to identify host genes involved in susceptibility to an infectious disease. These methods include: case control candidate gene association studies, genome-wide association studies and family-based, genome-wide linkage studies. There is substantial evidence from epidemiological, segregation and twin studies that host genetic factors contribute to susceptibility to leprosy (Abel and Demenais 1988; Feitosa et al. 1995; Haile et al. 1985; Wagener et al. 1988). Several genes that modulate cell-mediated immunity have been investigated and some appear to have a role in either susceptibility to leprosy per se, or to leprosy type (Fitness et al. 2002). Genome-wide association studies have made substantial advances in understanding of the genetic basis of many complex traits (McCarthy et al. 2008). Recently, two genome-wide association studies (GWAS) have been conducted to identify leprosy risk factors. In the first GWAS of leprosy, variants across six genes HLA-DR-DQ, RIPK2, TNFSF15, CCDC122, C13orf31 and NOD2 were shown to be associated with leprosy. Significant heterogeneity was detected across leprosy subtypes (multibacillary versus paucibacillary) for five SNPs across four genes RIPK2, C13orf31, NOD2 and LRRK2 with stronger evidence of association with the multibacillary subtype (Zhang et al. 2009). Second GWAS was conducted by Wong et al, 2010 in which they observed consistent associations between genetic variants in both TLR1 and the HLA-DRB1/DQA1 regions with susceptibility to Page 3

4 leprosy (Wong et al. 2010). The role of HLA linked loci in controlling leprosy and its subtypes is consistently supported by other studies also (Alcais et al. 2007; Ali et al. 2012; Alter et al. 2011; Fitness et al. 2002; Mira et al. 2003; Vanderborght et al. 2007). Genome wide linkage scans for leprosy susceptibility loci have also been conducted. Regions on chromosome 6q (Alter et al. 2012; Mira et al. 2004; Mira et al. 2003), 10p (Siddiqui et al. 2001) and 20p (Tosh et al. 2002) were identified and suggested to harbor genes affecting susceptibility to leprosy per se or to paucibacillary (PB) leprosy in particular. The susceptibility to leprosy and to different clinical phenotypes of the disease suggests that the nature of leprosy pathogenesis is apparently under a two stage genetic control whereby some genes affect intrinsic susceptibility to leprosy (i.e advancement from innocuous infection or carrier status to clinically evident disease) and other loci modify the clinical form of the disease (Mira et al. 2003). Although it is encouraging that attempts have been made to identify genetic loci which associate with susceptibility to leprosy and its subtypes at both candidate gene and genome-wide level; however the number of candidates showing consistent replication across populations are few. Among those that have been validated, genes with a potential or an involvement in demonstrated biological mechanism, form an even smaller subset. The ultimate aim of genetic research is to translate findings of GWAS and candidate gene studies into clinical care (Figure 1.2). Hence, there is a need to identify additional genetic variants and mechanisms which influence the disease outcome. India has a multitude of genetically diverse population groups. Though Indian populations seem to be ethnically and linguistically isolated, they are highly admixed through recent invasions. Due to myriads of genetic, geographic and socio-economic reasons, India carries majority of the global burden of infectious diseases such as leprosy. These facts make India a fertile ground for gene-hunting and validation of results from genome-wide association and candidate gene studies. Although many candidate gene association studies have been conducted to identify leprosy risk factors; where results suggests a polygenic nature of the disease with a high degree of heterogeneity among different populations. However, only a few results have been replicated unequivocally in different population groups. The present case control association study of polymorphisms in the regulatory region of the PARK2 and PACRG gene was carried out to genetically dissect the basic elements involved in the interplay between M. leprae and its human host. Since, leprosy is a complex disease caused by multiple genes, it was considered pertinent to explore gene gene interactions between the studied variations of the candidate genes in view of the polygenic nature of the disease. Page 4

5 Figure 1.2: Clinical translations of findings from numerous methodologies adopted in genetic research. There are two principal routes through which such translation might be effected. In the first, identification of novel causal pathways provides new opportunities for clinical advances of generic benefit to all those suffering from (or at risk of) the disease concerned. The second translational route lies through using knowledge of individual patterns of disease predisposition to develop more personalized approaches to disease management (McCarthy et al. 2008). Page 5