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The only methods of observing the immune system in archaeological populations is through analysis of pathological conditions manifested in the skeleton, and molecular analysis. The first establishes the presence of pathological conditions within a given population, and can provide insight into morbidity and mortality. However, the presence or absence of skeletal lesions cannot accurately reflect the ability of the individual's immune system. The immune system in anthropology is observed through the analysis of various forms of ancient DNA: microbial, nuclear and mitochondrial. The following chapter introduces how the human immune system functions in general, the function of cytokines and the overall response to tuberculosis (TB). The second half of the chapter introduces the various ways molecular anthropology can be utilised to study arcaheological populations. In particular the chapter discuses how the various forms of DNA can be utilised to establish the basic tenets of this thesis: confirm Inuit ancestry, analyze cytokine genotypes, and confirm or refute presence of TB. By determining cytokine genotypes it is possible to assess how the population may respond to infection by M. tuberculosis, which in turn could establish the reason for discrepancies between current TB rates of infection in Inuit, First Nations and non-aboriginal Canadian populations.
Tuberculosis (TB) remains a global health problem, with the World Health Organization (WHO) estimating that one third of the world's population is recovering from or infected with tuberculosis (World Health Organization 2010). Socio-economic factors influencing TB are commonly researched and acknowledged as factors in the disproportionate prevalence of infection amongst nations and ethnicities, and a growing interest has been expressed in environmental and biological factors that may have impact on a host's immunity and the severity of tuberculosis infection.
Compared to many global communities and nations the overall incidence rate  and the pulmonary TB rate in Canada are amongst the lowest globally (2/100 000 vs. e.g. Swaziland 510/100 000, World Health Organization 2011). However, within the Canadian population the Inuit maintain the highest incidence rates found within Canada as a whole, and often within individual provinces (Public Health Agency of Canada 2011). In a ten year period the Nunavut territory has maintained a rate multiple times higher than any other province or territory in Canada, with an incidence rate ( 174/100 000 in 2009) comparable to some of the most highly affected countries worldwide (Public Health Agency of Canada 2011). Provinces with high First Nations' populations and a high Inuit population in particular, such as Nunavut, the Northwest Territories, Manitoba and Newfoundland and Labrador sustain the highest overall incidence rates, and the highest prevalence rates for the Inuit population (Public Health Agency of Canada 2011).
Historically Inuit and First Nations' populations displayed vulnerability towards tuberculosis infection (Davies 1967; Ferguson 1950), and in the first half of the twentieth century demonstrated the highest incidence and mortality rates (Koch et al. 2008). Today, the Inuit have an incidence rate almost 6x higher than First Nations' (155.8 vs. 27.4), approximately 21x than that of the Métis (7.3), and 33x higher than the national average (4.7) (Public Health Agency of Canada 2011). While socio-economic conditions such as over-crowding, housing conditions, malnutrition, and inaccessible health care impact the incidence rates, growing academic and medical consideration has been given to the role that immunogenetics may have on resistance and/or susceptibility to tuberculosis infection. By examining observed immunogenetic diversity anthropologists can infer possible relationships between populations, estimate migration times and patterns and evaluate whether genetic diversity is a response to the selective pressures of a specific environment (Hill et al. 1999; Sanchez-Mazas 2001). In recent decades anthropologists and geneticists have explored a number of immunity related genes (ex. HLA, KIR, cytokine single nucleotide polymorphisms) in an attempt to explain health differentials and population based differences in disease incidence and prevalence (Sanchez-Mazas 2001; Larcombe 2005, Larcombe et al. 2008; Rempel et al. 2011). Various levels of the human immune system are responsible for combating pathogenic conditions and variability can be found in any number of genes therein.
The Human Immune System
At a cellular level the human immune response is designed to differentiate between self and non-self (Coico and Sunshine 2009). However, there are four main tasks required to fulfill the proper functioning of the immune system as a whole. The first is the ability for the immune system to recognize foreign intrusion into the body, referred to as immunological recognition; this initial function is provided by the innate system which provides an immediate response through the white blood cells and later by the lymphocytes of the adaptive system. Secondly, the "intruders" must be quarantined or eradicated from the system; this demands the response of immune effector functions, which often work in concert with each other and may consist of a host of different cell types: the complement system of blood proteins, antibodies, and the destructive capabilities of lymphocytes and other white blood cells. However, the immune system does not operate singularly, and must be controlled in order to prevent destruction of healthy cells, which can occur in individuals suffering from autoimmune diseases such as rheumatoid arthritis and lupus. This third property is referred to as immune regulation, or self-regulation; the lack of this ability to control immune response leads to the aforementioned auto-immune diseases as well as allergies and other detrimental conditions. The final task of the immune system is one of the primary functions of the adaptive portion of the immune system, that of immunological memory. This allows an individual to elicit an immediate and progressively stronger response with each successive exposure to a pathogen, what is known as protective immunity. However, there are many pathogens to which the immune system never develops a long-lasting immunity; this is the major concern of many health researchers who study complex diseases (Murphy et al. 2008).
The human body is varied in its actions and responses, between individuals and between populations. Much of the variation depends upon the history of the population, pathogens and environments encountered and experienced throughout the evolution of its inhabitants. The responses afforded by the human immune system against infection of any form are part of immune response. A major aspect of immune diversity is the Human Leukocyte Antigen (HLA) complex of genes in the major histocompatibility complex (MHC). HLA genes are important in the development of activated T-cells and their associated response to foreign microbes. Diversity comes from the over 200 highly mutable possible alleles (Eren and Travers 2000) in the HLA complex which are categorized into two classes I (-A, -B, -C) and II (HLA-DRB1, -DQA1, -DQB1, -DPA1, -DPB1) (Solberg et al. 2008). HLA class I and class II proteins are highly polymorphic and are essential in distinguishing self and non-self during immune response (de Bakker et al. 2006).
Hundreds of HLA alleles have been observed globally, and many with relatively high frequencies, exist within any given population. This diversity makes the collective HLA loci the most polymorphic in the human genome (Eren and Travers 2000). The combination of alleles on a chromosome is referred to as a haplotype (Murphy et al. 2008); a number of HLA haplotypes have developed amongst various populations. HLA diversity can often be utilised as a tool for assessing the relationship between populations. Anthropologists have used the distribution of HLA haplotypes to investigate human genetic relationships, reconstruct human migration patterns (Bodmer et al. 1997; Clayton et al. 1997), and assess genetic admixture and/or isolation in New World populations (Blanco-Gelaz et al. 2001). The role of the HLA complex is to provide individuals with protection against pathogens that invade the body and there is a direct link between a population's disease history and evolution and its HLA composition (Hill 1998). The HLA complex is responsive to myriad pathogens and their changeable nature provides immunological protection through various selective pressures, often related to human biological and cultural behaviours (Blanco-Gelaz et al. 2001). HLA haplotypes, of which there are many, are partially responsible for a population's ability to resist and combat different pathogens (Hill 1998). The frequency of these alleles can differ greatly between individuals and populations.
Innate Immune System
The defences of the human immune system range from physical barriers such as our skin and mucosal membranes to highly sophisticated response systems. Physical and chemical barriers are the frontline defences against foreign body intrusion. Most foreign organisms cannot penetrate skin unless it is damaged; some microorganisms enter through sebaceous glands and hair follicles but are initially combated by the presence of sweat, sebaceous secretions, fatty acids and hydrolytic enzymes (Coico and Sunshine 2009). The more sophisticated systems are activated after chemical and physical barriers have been penetrated and include a number of intracellular and extracellular mechanisms. The myeloid lineage comprises most cells in the innate immune system, including macrophages, granulocytes, mast cells, neutrophils, eosinophils, basophils, and dendritic cells. Some of these cells coordinate immune responses, induce inflammation, secrete signalling proteins for other cell activation and recruitment, use anti-microbial agents to destroy microorganisms, defend against parasites, and scavenge within the body and eradicate other dead cells. The lymphoid lineage matures in the bone marrow or thymus and then collects in lymphoid tissues throughout the body, and are found in large numbers in lymphocyte congregates known as lymphoid tissues and organs (Murphy et al. 2008). Mucous membranes and the skin are the first defence against invasion; foreign cells that prevail and break through these barriers are met by cells of the myeloid and lymphoid lineages.
The innate immune system is the dominant immune system in plants, fungi, and insects and in primitive multicellular organisms. It is thought to represent an evolutionarily older defence mechanism than the remainder of immune responses in modern populations. It is found in all orders of animals and plants as the immediate or general defence mechanism that responds instantaneously to changes in the host with a multitude of responses, but lacks specificity. Unlike the adaptive immune system, the innate does not confer lifelong immunity or the ability to respond accordingly with each successive infection (Alberts et al. 2002) however it is the mechanism that provides the initial discrimination between the self and the other.
The immune system is regulated by soluble mediators known as cytokines (Coico and Sunshine 2009). "Cytokine" is the general term for any protein that is secreted by immune cells in both the innate and adaptive systems (Coico and Sunshine 2009) and affects the behaviour of other cells with complimentary receptors. A subgroup of cytokines known as chemokines, are secreted proteins that attract cells with complimentary receptors from the rest of the body to the infected or foreign cells. It is through the release of cytokines and chemokines that the primary response of the innate immune system, inflammation, is triggered. Most infectious agents activate the innate immune system initially and induce an inflammatory response, which is usually generated in the early stages of immune defence. Inflammation is stimulated by cytokines released by affected cells and results in localised heat, swelling, and redness, all of which serve to establish a physical barrier against the spread of infection, and promote healing in affected tissue once the injury has been resolved (Stvrtinova et al. 1995). Inflammation also results in additional beneficial responses, particularly the attraction of neutrophils which alert the immune system to injury or infection and summon other immune cells such as leukocytes and lymphocytes. When stimulated by bacteria macrophages (a specific type of leukocyte) produce cytokines; these include tumour necrosis factor and interleukin 1 (IL-1) interleukin 6 (IL-6), TNFÎ±, Interferon gamma (IFNÎ³), and IFN-beta (IFNÎ²) (Lotze and Tracey 2005). IL-6, IL-1, and INFÎ± initiate a wide spectrum of biologic activities that help coordinate the host's responses to infection by microbial and viral pathogens. Most commonly they induce fever, which is why they are sometimes referred to as endogenous pyrogens (Roth and DeSouza 2001; Kluger 1991; Jansky et al. 1995; Roth et al. 1993; Lemay et al. 1990; Coico and Sunshine 2009).
Adaptive Immune System
The adaptive immune response is based on the ability to generate a vast assortment of antigen receptor specifications, cells that can identify the specific pathogen or foreign antigen present in the body (Medzhitov and Janeway 1998). The adaptive system has a number of mechanisms to combat infection; the main method is by specialized white blood cells known as lymphoctes (B cells and T cells) which can recognize and target pathogenic micro-organisms or infected cells (Murphy et al. 2008). However the ability and function of the adaptive immune system is predicated by the presence of the innate system. The innate system provides the foundation of immunological responses that are necessary for the proper functioning of the immune system. The adaptive immune response necessitates its presence, without the innate system the adaptive system would not function or exist. Likewise, the adaptive immune system is not activated for regulatory problems or to heal cuts and bruises, instead appropriate activation only occurs in response to a foreign pathogen (Medzhitov and Janeway 1998).
If the infection circumvents the innate immune response or persists for an extended period of time then the adaptive (specific) immune response takes over. Once a pathogen has been introduced to specialized B-cells and T-cells these cells are "educated" to remember the pathogen as a threat in the event of future infections. This is called immunological memory, a phenomenon which bestows lifelong protective immunity to the individual. An example of this type of protective immunity is the resistance to re-infection to chicken pox after the initial infection. This response and adaptation is particular to the individual and to each distinctive pathogen.
Genetic background plays a key role in determining the quality of cytokine response. The type of T helper cell response is a major factor in the production of key cytokines. A Th1 or Th2 response can affect the host's ability to eradicate an infectious agent (Romagnani 1996; Hurtado et al. 2003; Larcombe et al. 2005; Wilbur and Buikstra 2006). Th1 and Th2 responses are also linked to the system's response to external and internal stimuli. Normative secretion of a cytokine is increased in response to intrusion by a particular foreign element. Suppression, down regulation or an increase in production is continually affected by the secretion and production of a number of other immune cytokines promoted by the two branches of the T helper response (Romagnani 1996, 1999; Flynn and Chan 2001; North and Jung 2004) and individual genotypic profiles will affect these mechanisms of Th1 and Th2 responses.
Th1 vs. Th2
T helper lymphocytes or T cells can be divided into two unique subsets of immune cells based on their function and associated profile of cytokines (Romagnani 1996, 1999; Constant and Bottomly 1997; Flynn and Chan 2001; North and Jung 2004). The two subsets are referred to as either Th1 or Th2 immune responses; both develop from naive T cells and are activated dependent upon what type of microbial antigens t have been introduced to the host (North and Jung 2004). A Th1 immune response and associated cytokines are activated and produced in response to intracellular bacteria and some viruses (Romagnani 1999), such as tuberculosis or influenza, while Th2 immune responses and associated cytokines are stimulated by complex parasites such as gastrointestinal nematodes (Romagnani 1999).
Th2 helper cells promote the production of cytokine responsible for strong antibody production, eosinophil activation and inhibition of several macrophage functions, thus providing a phagocyte-independent protective response. Cytokines such as IL-4, IL-5, IL-6, IL-10 and IL-13 are associated with the Th2 or humoral form of immune response (Romagnani 1999). Th1 type helper cells produce a specific contingent of cytokines typified by the production of interleukin (IL) 2, interferon gamma (IFNÎ³) (Surcel et al. 1994) and tumour necrosis factor beta (TNFÎ²) (Romagnani 1999). These cytokines are generally considered pro-inflammatory cytokines and are involved in cell-mediated immunity. However, a number of functional reactions are instigated by a Th1 immune response including: the production of opsonizing  and complement-fixing antibodies, macrophage activation, antibody-dependent cell cytoxicity, and delayed type hypersensitivity (DTH) (Mosmann and Coffman 1989; Romagnani 1994, 1996, 1999). Key to an immune response to tuberculosis infection is the ability to stimulate phagocytosis  and destruction of microbial pathogens. The polarised nature of Th1 and Th2 cells creates a greater significance on the phenotype of the host's immunogenetic profile. The host's differential production of a wide variety of cytokines is often dependent upon the SNPs that affect the gene and function responsible for that cytokine concomitantly to alleles for all other cytokines. SNPs in the promoter region of a cytokine create a cytokine genotype. The association of SNPs in a number of cytokine promoter regions associated with disease prognosis can produce the overall ability for a host to actively and effectively combat foreign intrusion.
Both innate and adaptive immune responses depend on what was previously described as white blood cells, or leukocytes. Leukocytes are an unrestricted immune cell that combats abnormalities in the body (Murphy et al. 2008). However there is a vast range of cells that are produced by the immune organs, as well as by cells which identify a pathogen and signal for the presence of a specific combative cell. Some of the cells that combat and signal are known as chemokines and cytokines. Cytokines are small proteins, usually around 25 kiladaltons that are released by various cells in the body, usually in response to stimulus from other cells within the immune system (Murphy et al. 2008). There are two major lineages or families of cytokines, the haematopoietic and the TNF family. The haematopoietic family includes growth hormones and interleukins with roles in both the innate and adaptive immune systems. The TNF family also functions in both immune response systems. The importance of identifying cytokine polymorphisms in association with certain diseases, like tuberculosis can lead to a better understanding of disease pathology, identification of potential markers of susceptibility, severity and clinical outcome, identification of potential markers for responders vs. non-responders in clinical and therapeutic trials, identification of novel strains, either extinct or extant, and strategies to prevent diseases or improve existing preventative measures, such as vaccines (Bidwell et al. 1999). There are obvious differences in the implications of studying polymorphisms in ancient populations compared to modern living populations. However, understanding the way a population combated disease at the immunological level can reveal important factors about the evolution of the human immune system, as well as differences between individual and population expression of cytokines.
Cytokine polymorphisms are common immune modifiers (Wagner et al. 1999); however they are rarely linked to disease causation, except in a limited number of isolated cases. Instead cytokine polymorphisms act as disease modifiers by affecting disease severity, the ability to effectively combatdisease and mount an immune response, and in some cases increased or reduced susceptibility to infection. Cytokine SNPs most commonly affect disease outcome in inflammatory, allergic, autoimmune or immunodeficiency diseases. Cytokine SNPs have also been linked to transplant outcome, as individuals with certain polymorphisms may be more likely reject or accept an organ based on their cytokine genotype profile (Turner et al. 1997; Asderakis et al. 2001). Chemokine and cytokine SNPs all demonstrate variability between ethnic populations (Borman et al. 2004; Rovin et al. 1999; Hoffman et al. 2002; Kaur et al. 2007).
This research analyzes associations between cytokine polymorphisms and common complex diseases (TB), with particular emphasis placed on associations between cytokine polymorphisms in the promoter regions of the genes.
Cytokine Polymorphisms and Disease
A number of associations have been made between cytokine polymorphisms and a number of pathological conditions (for a summary, see Bidwell et al. 1999); because cytokines are not only important mediators of the immune system but also of inflammatory response, their impact on a number of conditions is significant. Research has identified linkages between specific cytokine polymorphisms and cardiovascular diseases, cancers, neurodegenerative disorders, periodontal disease, immune-mediated diseases such as allergic asthma and other autoimmune diseases, and transplant rejection or acceptance (Hollegaard and Bidwell 2006).
Allelic variants of cytokine genes are associated with higher or lower production of cytokines both in vivo and in vitro (Perrey et al. 1998). The polymorphisms studied in this research are all biallelic, meaning single genes with two alleles present; the genotype is expressed as homogenous for either A, T, C, or G, or heterogeneous. The genotype is linked, to a high, low or intermediate classification of cytokine production based on the pair of nucleotides represented at the locus (see Table 3.1). Important polymorphisms reside in the promoter regions of IL-10, TNFÎ±, IL-6 and the first intron of IFNÎ³ (Perrey et al. 1998). The cytokine SNPs analysed and therefore important to this dissertation are IL-6 (-174) (rs1800795) (C/G), IL-10 (-1082) (rs1800896) (A/G), IFNÎ³ (+874) (rs3087456), and TNFÎ± (-308).
Table 3.1 Cytokine Genotypes and Associated Phenotypes
(modified from Larcombe et al 2005)
Phenotypic Affect (production)
IFNÎ³ is a 143 amino acid homodimer of the interferon family and is a key Th1 pro-inflammatory cytokine (Miyazoe et al. 2002; Gonsalkorale et al. 2003) produced primarily by T cells and natural killer cells (Tso et al. 2005) and has pleiotropic effects in immunoregulation and inflammation dependent upon context. The effects of the IFNÎ³ or IFNÎ³ receptor's knock out results in decreased resistance to bacterial infection and the effects of tumours (Murphy et al. 2008). Macrophage activation by IFNÎ³ is essential for adaptive immunity, which plays a large part in the ability of the adaptive immune system to protect the body (Lopez-Maderuelo et al. 2003). This is also a major function of the cytokine in combating and protecting the body against specific pathogens such as Mycobacterium tuberculosis (MTB or TB) (Tso et al. 2005). The basic pathway of immunity to many mycobacteria involves the production of IFN-g, IL-12 and IL-18 by the infected macrophage. This occurrence is probably confined to early infection and is required to establish a Th1 response. Th1 cells secrete IFN-g and TNF-a, which activate macrophages to kill mycobacteria via mechanisms involving Nitric Oxide production (Flynn 1999; Murray 1999). The specific polymorphism of interest in many tuberculosis-associated studies is IFNÎ³ (+874). Many populations which maintain high levels of TB prevalence and incidence express higher frequencies of the A/A genotype compared to the T/A and T/T genotypes. IFNÎ³ low producers (A/A) are therefore at a higher risk of developing TB (Tso et al. 2005).
Interleukin 6 (IL-6) is a multifunctional (Akira et al. 1990) inflammatory cytokine (Fishman et al. 1998) whose major function is antibody induction (Akira et al. 1990) and it reinforces the differentiation of CD4 cells into Th2 cells. The lack of regulation of IL-6 can lead to autoimmune diseases and disorders. IL-6 is found in large amounts, and is produced by the synovial tissue in patients with rheumatoid arthritis (Houssiau et al. 1988). Similar to TNFÎ± it has both systemic and localised effects on both the innate and adaptive immune responses. TNFÎ± is a protein produced by macrophages and other immune cells (Tracey 1994). It is a Th1 pro-inflammatory cytokine (Miyazoe et al. 2002; Gonsalkorale et al. 2003). At the local level TNFÎ± activates vascular endothelium and increases vascular permeability, which leads to increased entry of IgG, complement, and cells to tissues and increased fluid drainage to lymph nodes. At the systemic level, TNFÎ± produces fever and shock and mobilizes metabolites. It is one of the few cytokines directly associated with disease causation (Yucesoy and Luster 2007). TNFÎ± receptor polymorphisms have been directly linked to periodic fevers (Aganna et al. 2001; Aksentijevich et al. 2001; Leonard 2001)andit is produced in response to bacterial presence, including tuberculosis bacilli. The polymorphism in the promoter region of the gene that encodes for TNFÎ± at nucleotide position -308 is associated with a number of diseases (Van Dullemen et al.., 1995; Present et al.., 1999; Yucesoy et al. 2001). In some cases the association results in an improved outcome, in others the association of TNF alpha (-308) is pathogenic (ex. Rheumatoid arthritis, see Sander and Rau, 1997; Feldman and Brennan 2001).Interleukin-10 is an anti-inflammatory Th2 cytokine (Tso et al. 2005; Gonsalkorale et al. 2003) and considered a potent inhibitor of most Th1 effectors. It down regulates the IFNÎ³ production of T cells, TNF, and nitric oxide (Tso et al. 2005). Research has discovered that in some populations there is no association of IL-10 with either an increased or decreased TB susceptibility (Tso et al. 2005), however in mice models over expression potentially increases chances of reactivation of latent TB (Tso et al. 2005). Il-10 can be highly toxic and has been implicated as a lethal mediator of acute and chronic infection (Tracey 1994). There is a weak association between IL-10 and IFNÎ³ with relapse and extrapulmonary TB (Tso et al. 2005).
Canadian Aboriginal and Inuit Specific Immune Diversity
Immunological diversity in Canadian First Nations and Inuit populations has been observed for several immunological characteristics. Amongst them cytokine SNP diversity and Killer Immunoglobulin-like Receptors (KIRs), which are largely responsible for regulating the function of natural killer cells and some T-cell subsets, thus affecting both innate and adaptive immune responses (Rempel et al. 2011). The KIR cluster contains 15 genes and 2 pseudogenes, 6 of which are segregated within the centromeric and telomeric portions of the cluster. Significant differences have been observed in Canadian aboriginal populations in comparison to control (Caucasian) populations for a number of genes within this KIR cluster. Canadian aboriginal population's KIR cluster demonstrates a greater immune activating phenotype associated with genes of the B haplotype situated within the telomeric region (Rempel et al. 2011).
Immunity and Tuberculosis
The disease tuberculosis is spread by multiple strains of mycobacteria including, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium canetti. There are also strains of mycobacterium that cause other diseases, such as Mycobacterium leprae and Mycobacterium avium neither of which result in the pathogenesis of tuberculosis. Tuberculosis is caused by the pathogen Mycobacterium tuberculosis, an aerobic intracellular pathogen that can survive and multiply inside macrophages and other human cells. It has evolved to avoid destruction by innate and adaptive mechanisms of immunity in certain immunocompetent humans. This allows TB to survive at the infectious stage for a prolonged period of time (North and Jung 2003). Most human populations are resistant because of the ability to generate an effective immune response; of those who are infected between 70-90% managed to prevent the infection from becoming established in the host (North and Jung 2003). Those who develop an active pathological condition are treated with specific antibiotics to reduce the bacilli in the host.
No pharmaceutical treatment for tuberculosis infection was available prior to the mid twentieth century and control of the infection was limited; the discovery of antibiotics such as streptomycin in the 1940's led to a continual decrease in TB prevalence for almost 40 years. In the 1980's a global resurgence in TB cases, coincided with the onset of HIV/AIDS epidemics and shortly after multi-drug resistant strains of TB (U.S. Department of Health and Human Services 2008). Now TB bacilli comes in many forms, either mono-resistant or poly-resistant, which refers to resistance to either one or multiple first line TB drugs (isoniazid or rifampin) (U.S. Department of Health and Human Services 2008). Extensive drug resistance TB (XDR TB) strains are those that are resistant to both isoniazid and rifampin, plus any fluoroquinolone and at least one of three injectable second line drugs (amikacin, kanamycin, or capreomycin) (Shah et al. 2007; U.S. Department of Health and Human Services 2008). Similarly, conditions that involve immune suppression or immune compromised hosts, such as HIV/AIDS, have led to systemic tuberculosis which spreads to many of the other major organs (North and Jung 2003; Shafer et al. 1991) suggesting that even in highly susceptible and immune system vulnerable humans there are levels of immune competence. This assumption is based on the premise that a systemic mechanism works in defence of the infection to sequesterthe infection in the lungs and no other major organ. This is in part due to the importance of Th1 immunity which not only protects those resistant to infection through its complement of cytokines and associated immune cell responses and functions, but also enables a portion of the immune competent susceptible human with active disease to create a growth-restricting influence on Mtb and its pathology (North and Jung 2004).
The protective and immunological response to tuberculosis is a complex and layered system, involving many different aspects of the human immune system, which once activated is generally successful in containing the active pathogen, but not eradicating the infection itself (Flynn and Chan 2001). Resistance to tuberculosis depends critically on antigen-specific T-cell mediated activation of macrophages  (Surcel et al. 1994). Macrophages, acting as antimycobacterial effector cells, mediate killing or inhibition of bacterial pathogens, although the exact mechanism is still debated. In the case of M. tuberculosis, the infection persists within granulomas in the organs of infected hosts (Flynn and Chan 2001), thus macrophages encase an active infection reducing it to a latent stage. The granuloma consists of macrophages and giant cells, T cells, B cells and fibroblasts. One of the key endogamous activating agents that triggers the antimycobacterial nature of macrophages is interferon gamma (IFNÎ³) (Lurie 1942; Suter 1952; Mackaness 1969; Flynn and Chan 2001)
In an organism with a properly functioning immune system it is possible to be infected with TB and not manifest the disease. The "disease" develops in individuals whose immune system cannot control the bacilli present after infection. The development can occur immediately or latently. Only 10% of those whose immune system functions properly, and who are infected with TB will subsequently develop active disease (Hoal 2002). Activation of disease might occur later in life, when the immune system has a weakened ability to contain and isolate the bacilli or if the immune system is compromised by another disease such as HIV/AIDs (Hoal 2002). The innate immune response at the time of infection is crucial for the initial isolation, identification and containment of the bacilli. The immune response to tuberculosis infection involves a complex network of cells and a cascade of soluble mediators. This response determines whether the bacilli are eradicated, or take residence within their macrophage niche environment resulting in active infection (Hoal 2002).
There are many different Mycobacteria strains, those that result in the disease often differ in the immune response invoked; this can result in differences in macrophage growth (Hoal-van Helden et al. 2001a), cytokine release (Hoal-van Helden et al. 2001b), efficiency in person to person transmission, and the susceptibility of the host to infection (Hoal 2002). The strains of mycobacteria which cause tuberculosis dictate the genes that will be associated with disease pathology; ergo the same pathogen may evoke a differing cytokine production dependent upon the individual and the population, reinforcing the linkage between ethnicity, immune response and tuberculosis infection. Likewise animal models have demonstrated that, genetic resistance or susceptibility can be selected for within a population (Hoal 2002).
Population-based immunological variation
The degree of immunological diversity that exists between ethnic groups and individuals is apparent in differences observed in blood group analyses, the distribution of the human leukocyte antigen complex (HLA), killer cell immunoglobulin-like receptors (KIR), and cytokine SNPS. The immune response available to a population is highly linked to its evolutionary history. Different populations have coevolved with different pathogens and pathogen loads, have been exposed to epidemic or endemic levels of diseases and had their gene pool adversely or beneficially affected. A major impact on the ability of a population to mount an effective immune response is its historical interaction with a pathogen. For example, "crowd diseases", those that developed with the urbanization and increased population density of cities (Diamond 2002) have existed for a longer period of time in the Old World, where domestication and city states first arose (Lipsitch and Sousa 2002; MacNeill 1998). In the Old World, populations spent a longer time living in continuous close proximity and exchanging not only a wide range of pathogens, but also genetic information through admixture. These populationslikely encountered a wide range of diseases and conditions over extended periods, which allowed them to develop an efficient adaptive immune response, while eliminating individuals who were highly susceptible. Similar to the Darwinian theory of selective fitness, individuals who succumb to a pathological condition, especially those of a young age, will be unlikely to pass on the genetic information that makes them vulnerable. In the meantime, those within the population who are able to elicit an effective immune response can potentially pass their genes on to successive generations and the population develops a level of "herd immunity".
New World populations experienced a contrasting pathogen encounter. Crowd disease developed later in the New World compared to the Old World, possibly making the populace more vulnerable to novel diseases carried with colonists. New World aboriginal populations developed immune responses prior to migration into the New World. Therefore part of their immunological profile is shared with Old World populations. As mentioned above, many New World aboriginal populations experience immunological characteristics thought to be directly related to the pathogen load of their individual environments (Hurtado et al. 2003; Wilbur and Buikstra 2006). This translates into a propensity for Th2 responses in New World populations, those better suited to fighting parasitic and fungal infections (Hurtado et al. 2003), but vulnerable to viral and bacterial infections. In contrast, Caucasian (and other Old World populations) developed a propensity for Th1 responses in Caucasian (Old World), providing better immunity against bacterial and viral pathogens and a vulnerability to foreign parasites and fungal infections (Wilbur and Buikstra 2006).
Researchers have concluded that populations who share similar ethnicity (biological and cultural history) exhibit shared variable responses and rates of infection (Larcombe et al. 2005; Ducati et al. 2006). This condition would not be independent of socio-economic and socio-cultural factors, but exacerbated by any detrimental aspects. Similarly, environment, including host-pathogen interaction and pathogen load of past populations, cannot solely explain current populations' immunological response (Rempel et al. 2011). However, understanding the various forms of immune diversity present within a population can shed light on the population's susceptibility or immunity to various pathological conditions (Rempel et al. 2011). Research comparing the genetic structure of the host population (Ewald 1994 for example), suggests that potential hosts within an ethnic population may express a wide variety of genetic characteristics affecting susceptibility and resistance. A large population with appreciable genetic variety maintains the ability to better combat infection by a pathogen. The main reason for this is that a pathogen may encounter a number of individuals with an immune profile able to effectively combat infection before encountering a viable host (Wilbur and Buikstra 2006). Many pathogens therefore never become highly virulent in these populations but are forced into fairly benign forms so that they do not decimate the population and self-eradicate (Wilbur and Buikstra 2006). In a population lacking in genetic variability of the immune response, the pathogen need not evolve to a less virulent form, it can freely infect and multiply without decimating a population. Evidence indicates that Native American populations are more genetically homogenous at many loci including those that affect the immune system and response to TB (Wilbur 2005). The immunogenetic variability of Native populations, both Arctic and southern, was likely reduced by genetic bottleneck events, war, poverty, starvation, and previous epidemic infection, possibly leading to a more vulnerable population (Wilbur and Buikstra 2006).
Wilbur and Buikstra (2006) argue that a number of factors can contribute to the effect of an infection within a population; social and cultural disruption such as the increase in population densities and widespread relocation of native groups would have altered susceptibility of native populations (Wilbur and Buikstra 2006). They suggest rather than assuming co-evolution of a host and pathogen would necessitate acquired immunity, that some populations may never gain an adequate immune response to certain pathogens. There is adequate evidence to support this supposition in Native South American groups; in certain populations individuals who have active TB or have been treated for TB are unable to mount and effective a Th1 response, and thus react as if they were immunologically "naÃ¯ve" to the pathogen (Wilbur 2005; Wilbur and Buikstra 2006).
In North American aboriginal groups such as the Inuit, Eskimo, Tlingit, Tsimshiam and Na-Dene unique HLA haplotypes have been identified (Pablo et al. 2000). Amerindian populations also exhibit significantly reduced allelic and haplotypic HLA diversity when compared to populations from Africa, Europe and Asian. Amerindian populations are often clustered together and show less "genetic distance" between each other compared to all other populations (Tsuneto et al. 2003). When compared to Old World populations Amerindian groups often cluster together, but analyses sometimes reveal interesting linkages between populations (Pablo et al. 2000; Tsuneto et al. 2003).
Hoffmann and colleagues (2002) found a striking difference in allelic frequencies between American black and whites for IL-2 and IL-6. They also observed that Asian descent populations compared to Caucasians (referred to as whites) have a considerably higher proportion of genotypes that result in high IL-6 and low IL-10 and IFNÎ³ production. A different study saw a higher frequency of the down-regulating IL-10 (-819) T/T genotype and IL-10 (-1082) A/A genotype in Black American women compared to white American women; the genotypes were 3.5 times and 2.8 times more common in black women (Ness et al. 2004). The study observed that African-American women were more likely than Caucasian women to express allelic variants known to increase the expression of the pro-inflammatory cytokines and less likely to express allelic variants known to increase expression of the immune-system-suppressant cytokine IL-10 (Ness et al. 2004). Hispanic descent groups differed from Caucasians in the inheritance of the high IL-6 producing allele and a reduction in the inheritance of the high IL-10 polymorphic alleles (Hoffmann et al. 2002).
Ethnic populations worldwide exhibit varying immunogenetic expressions of the SNPs associated with TB immunity. In particular, the four cytokine SNP's analysed within this study have been linked with various clinical outcomes in a number of populations. . TNFÎ± (-308) is associated with TB susceptibility in India (Selvaraj et al. 2001) there appears to be no significant association between the same polymorphism and TB in Cambodia (Delgado et al. 2002). IL-10 SNPs were not associated with disease susceptibility in Gambian populations (Bellamy et al. 1998), however, a significant correlation was observed between the A/G genotype and TB disease in Cambodian populations (Degaldo et al. 2002). In China there is a high association between the A/A genotype of IFNÎ³ (+874) and TB infection (Tso et al. 2005). This genotype results in a low producer phenotype, which may impair activation of macrophages and result in an increased risk for the development of TB in individuals exposed to the bacilli. In this particular population the risk is calculated at 3.79 times higher than if the individuals had an intermediate or high production phenotype. The A/A genotype is related to TB severity and/or reactivation of the infection (Tso et al. 2005). Similarly in Sicilian populations the T/T genotype is associated with protective factors in TB development (Tso et al. 2005). Sicilian populations show a decreased frequency of the A/A genotype and an increased frequency of the T/T genotype in comparison to the Chinese populations, which shows a significant difference in frequencies (Tso et al. 2005).
No previous genetic research exists on the immune system of the Inuit in historic or pre-Contact periods. What information is available about the occurrence of infectious diseases and immune response in the Inuit is based on historic records such as hospital records, birth and death records, and doctors' notes and journals. Genetic analyses on contemporary Inuit populations focus on human leukocyte antigen (HLA) diversity, generally in comparison to other populations (Chu et al. 2001; Tsuneto et 2003), but there still remains a gap in the knowledge concerning the genetic effect of the immune system on disease progression in the Inuit as well as an understanding of the synergistic relationship between the Inuit and the arctic environment. Even given the fact that the highest rates of TB prevalence and incidence occur in Nunavut and northern Manitoba Inuit communities research has been focused thus far on Canadian non-aboriginal populations, primarily Caucasian, and Canadian First Nations reserve and non-reserve individuals. By examining the SNPs in promoter regions of specific cytokines for archaeological Inuit populations, and attempting to identify the genotypic and phenotypic profiles of these cytokines, inferences can be made about possible selective pressures of the arctic environment on the Inuit's immune system. Fluctuations in selective pressures and immune response can be estimated through the analysis of archaeological immune profiles in comparison to contemporary Inuit profiles as well as other contemporary populations Linkages can then be made between prevailing TB rates and the immune profiles of previous Inuit populations by examining the differences between not only longitudinal Inuit populations but other First Nations' populations and non-aboriginal Canadians.
In a recent study it was found that a Caucasian cohort maintained a high frequency of SNPs in the promoter regions of several TB associated cytokines (Larcombe et al. 2005; Larcombe et al. 2008). This led to the suggestion that this group appears to favour a Th1 immune response. In contrast a First Nations cohort as well as Filipino descent populations favoured a Th2 immune response (Larcombe et al. 2005; Larcombe et al. 2008) based on the high frequency of Th2 cytokine promoter SNPs. The Th1 response is characterised by the release of IFNÎ³ and TNFÎ± which have been proven to provide protection against TB infection (Kamijo et al. 1995) while the Th2 immune response is characterised by the production of cytokines, such as IL-4, IL-6 and IL-13, associated with parasitic and fungal infections (Marquet et al. 1999). Caucasians therefore maintain a higher frequency of TNFÎ± (-308) and IFNÎ³ (+874) allele SNPs, both of whose phenotypic expression is associated with enhanced production of their resultant cytokines (Louis et al. 1998; Wilson et al. 1997; Kroeger et al. 1997; Allen 1999). Aboriginal and Filipino descent populations examined within this study generally maintained a higher frequency of IL-6 (-174) allele and the associated high production phenotype. First Nations aboriginals examined also appear to maintain lower production phenotypes for IL-10 (-1082) compared to Caucasian populations. Realistically, given the manner in which the New World was populated, and the sharing of 5 haplogroups within North America, the likelihood of an association between First Nations and Inuit is high. Most likely the Inuit will present similar SNPs to those found in the First Nations groups of Canada, with allowances made for inter-tribal mutation, bottlenecking, and restricted gene flow. Two First Nations groups which can be utilised to illustrate the immune response in First Nations groups are the Dene and the Cree populations. The Dene have SNPs associated with low production of TNFÎ±, IFNÎ³, and IL-10, and high production of IL-6; the Cree are also low producers of TNFÎ± , but they may also exhibit SNPs associated with low to intermediate production of IFNÎ³ and IL-10 (Larcombe et al 2005; Larcombe 2005; Larcombe et al 2008). The osteological record has shown that parasitic and fungal infections, as well as malnutrition were common in the pre-Contact New World (Marquet et al. 1999). It has been shown that helmiths and macro-parasites have an impact on the Th2 immune response in New World aboriginal populations (Hurtado et al. 2003; Wilbur and Buikstra 2006). Some aboriginal populations produce large amounts of antibodies while healthy; this is thought to be a result of the stimulus of macro-parasites on the Th2 pathway (Hurtado et al. 2003; Wilbur and Buikstra 2006). Both the Dene and the Cree exhibited cytokine SNPs association with high production of IL-6 (Larcombe et al. 2005; Larcombe et al. 2008), which may be suggestive that First Nations and Inuit populations immune system adapted to the environment in which it developed.
The conclusion is that ethnic identity likely plays a role in the variable response and rate of infection in different populations (Larcombe et al. 2005; Ducati et al. 2006) regardless of disease. However cytokine expression, in particular IL-10, IL-6, IFNÎ³ and TNFÎ±, significantly impacts the clinical outcome of TB, as well as the vulnerability of a population to initial and re-infection by tuberculosis bacilli. Differing incidence rates for TB, like those seen within aboriginal populations in Canada, may depend on the duration of ancestral selection for resistance to TB (Tso et al. 2005), but more likely is an expression of environment, genetics and individual adaptation.
Ancient DNA and Anthropology
The first successful application of molecular analysis to human remains occurred in 1985 (Paabo 1985) and began a ``revolution`` in not only how we study past populations but how we understand them. Research that explores some of the same questions that are part of traditional anthropological research is still undertaken, but the methods used to answer them, and the depth of the information now available has increased exponentially. The application of ancient DNA (aDNA) research methods to anthropology and archaeology are unlimited, and the development of new technologies and methodologies is continuous. Initially aDNA studies focused more on information garnered from mitochondrial DNA (mtDNA) rather than nuclear DNA (DNA), this is directly related to the degraded and highly fragmented nature of aDNA. Unlike mtDNA, which is multi-copy in form, nuclear DNA is single copy in form and damage to the nuclear sequence is more likely to be problematic in comparison to mtDNA (Hummel 2003).
Traditional methods utilised by physical and biological anthropologists are the foundations to molecular studies; the analysis of pathogens requires that a researcher be able to ascertain a provisional diagnosis, locate a viable lesion that may contain pathogenic DNA, and also provide a differential diagnosis to argue that the destructive sampling procedures of aDNA analysis are plausible, required and valid. Likewise, cultural and familial affiliation should be assessed via archaeological survey of a burial; individual identification should be undertaken using traditional methods for aging, sexing, and health indicators. Constructing a palaeopathological inventory of skeletal remains is a reliable method for determining the overall health of an individual. Molecular analysis can confirm the presence or absence of a pathogen; however pathogen identification cannot indicate the severity of the infection, the extent of dissemination, or its interaction with nutritional and degenerative diseases. Molecular analysis should be undertaken only when a definitive conclusion cannot be drawn from the osteological record; DNA analysis is by its nature a destructive process. Despite the limitations and hazards of undertaking aDNA studies, the outcome of ancient DNA (aDNA) analyses is a greater understanding of a number of anthropological issues concerning the familial history of hominins, the peopling of the New World, the presence of pathogens in certain environments and basic demographic information. The ability to resolve these issues makes aDNA analyses invaluable despite the destructive quality of its methodologies and the somewhat prohibitive cost of its utilization.
The main source of DNA used in many laboratories for ancient investigations is bone or teeth (Keyser-Tracqui and Ludes 2005); soft tissue is also used, but differential preservation of interred remains sometimes precludes the availability of this particular tissue type. Multiple skeletal elements of a single individual are often found, resulting in the possibly of significant amounts of DNA-rich samples. Teeth which are represented in multiple copies are the ideal choice for sampling. The enamel encapsulated dental pulp of teeth provides a uniquely preserved source of DNA (Pfeiffer et al 1999). Regardless of environment, if the enamel is intact, lacking cracks and caries that have exposed the dental pulp to contamination and degradation, a rich source of well preserved, highly concentrated DNA is available. Other skeletal elements that are candidates for molecular sampling include ribs, which also appear in the skeleton as multiple copies. A single bone, rib, femur or humerus, can yield varying concentrations of DNA depending on the aspect sampled. This is generally due to varying rates of preservation and the environment in which the bone was preserved (Schultes et al. 1997; Hummel 2003). In pathogenic analysis it is sometimes required that direct sampling of a lesion occurs, to ensure pathogenic DNA is collected along with the endogamous human DNA. Regardless of the skeletal element chosen, the destructive process of retrieving aDNA permanently removes the sample from the osteological record.
The preservation and quality of skeletal remains, as well as the environment from which the remains were excavated and then later curated, impact the quality and concentration of the DNA. Preservation depends upon the coincidence of a number of environmental factors. In terms of archaeological preservation one of the most important aspects is the occurrence and amount of microorganisms in the soil and surrounding environment. Two additional factors that impact the quality of skeletal remains and by relation the quality of preserved DNA are that low pH levels restrict the formation of certain minerals that can completely destroy a skeleton (Herrmann and Newesley 1982) and low levels of alkaline and urea, usually casused by high levels of excrement in the soil, prevent inhibition and degradation of the skeletal material as well as encourage molecular stability and quality (Hummel 2003). A major factor for post excavation preservation during curation is the temperature at which skeletal remains are stored. Remains kept at room temperature result in the degradation of DNA quality in a relatively short period of time (Hummel 2003). Ergo, remains excavated from an environment with low pH levels, low alkaline and humic acid levels, cool or cold temperatures (Gilbert et al. 2004; Lindahl 1993), with low levels of microorganisms result in functional molecular samples.
Methodologies can also play an important role in the ability and viability of DNA amplification. One of the most crucial aspects of ancient DNA analysis is the extraction process. Mistakes at this level can lead to contamination, low concentration and even destruction of DNA. There are many protocols available, some are originals while many others have been adjusted over time with experimentation. There are four basic categories of extraction protocols: phenol, silica, boiling and chloroform. The phenol and silica based methods are those that are more widely used in contemporary research (Hummel 2003) and varying degrees of success for each methodology have been published (Lassen et al. 1994; Loffler et al. 1997; Yang et al. 1998; Hummel 2003). Each of these extraction methods may utilise a different sample preparation procedure (i.e. removal of cortical bone, powdering, washing solutions). Kit methods are primarily designed for modern and forensic DNA analyses and are not currently available for aDNA analyses. Mitochondrial DNA has been successfully extracted and amplified using all of these methods, while nuclear DNA is more successfully amplified via the silica and phenol methods (Hummel 2003).
The application of ancient DNA studies in anthropology has been used to address a wide range of topics. The study of the migration of early hominins and the structure of their populations through mitochondrial analysis has led to a better understanding of how groups migrated from Africa to populate the rest of the world (Kahn & Gibbons 1997; Ward & Stinger 1997; Hoss 2000). The ability to discriminate between individuals both forensically and anthropologically has led to changes in the methods in which the legal system carries out justice, which was based on methodologies created for the study of ancient DNA (Kaestle and Horsburgh 2002). Methodologies and technologies developed through analysis of aDNA are also applicable to topics peripherally associated with anthropology or that through the use of molecular methods have only recently become important to anthropological studies. Some of these areas include the examination of museum specimens for identification and conservation of faunal and floral remains, known as conservation biology and genetics (Hummel 2003; Spencer et al. 2010) the screening of genetically modified organisms in microbiology; the study of infectious diseases and their evolutionary record, in particular their interaction with the human species and the co-evolution in different temporal and geographic populations (O'Rourke et al. 2000; Hummel 2003).
Methods developed to analyse the immunogenetic profiles of past populations though the amplification of aDNA may eventually reveal links between historical, cultural, and biological events with contemporary health issues. The protocols in this study are an example of how these methodologies can be implemented in archaeological Inuit populations as foundations to further knowledge. A limited number of molecular studies examining immunogenetic profiles in past populations currently exist. The majority of comparative information is extrapolated from analyses of past Oji-Cree Canadian populations from Manitoba (Larcombe 2005) and from analyses of contemporary Canadian Dene, Caucasian and Cree populations (Larcombe et al. 2005; Larcombe et al. 2008). These studies provide a baseline of comparison not only for contemporary Inuit susceptibility and vulnerability, but for genotype frequencies fluctuations that may have occurred between different ecological niches, cultural groups, and temporal populations. By examining the genotype frequencies and immunogenetic profiles of past Inuit populations, it becomes possible to understand the effect of past selective pressures on contemporary health issues, such as the Inuit's disproportionately high susceptibility to tuberculosis.
Mitochondrial DNA (mtDNA) is a circular multicopy DNA found within the mitochondria of an organism's cell; there are multiple mitochondria containing up to 1000 copies of the same genetic information in each cell. In humans each mitochondria contains 16 570 base pairs, coding for a total of 37 genes (Hummel 2003). Unlike chromosomal nuclear DNA, mtDNA is only inherited through the female portion of the previous generation, and therefore represents a haplotype, as all members of a maternal lineage show the same mtDNA (Hummel 2003). Each ovum carries a complete copy of the whole mitochondrial genome; in contrast the sperm does not contribute the mitochondria it carries to the zygote. This process results in the transfer of only the maternal mitochondrial genetic information through the generations, and each member of the maternal line is a member of the same lineage and forms a haplogroup. Mitochondrial DNA does not recombine (Stoneking and Soodyall 1996; Mesa et al. 2000) and contains an abundance of polymorphisms in the non-coding regions (Bianchi et al. 1998) which make it ideal to study short group associations. The fact that mtDNA does not experience recombination means that identical copies are located throughout members of the same haplogroup, and differentiation occurs only through mutation, which occurs at a much higher rate than seen in nuclear DNA.
Hypervariable regions I and II (HVR I and HVR II), located on the D-loop control region of the mitochondrial genome, manifest the most sequence polymorphisms in mtDNA. In total this region spans 900 bp, of which 600 bp are the HVR I and HVR II regions (Hummel 2003). These regions reveal approximately 3% variability between individuals, and the associated polymorphisms often cluster around so-called "hotspots" (Hummel 2003: 23). This enables researchers to identify individuals at the family lineage level (i.e. Butler and Levin 1998; Schneider et al. 1999), but these "hotspots" are also valuable in the analysis of population genetics or phylogenetic studies (Boles et al. 1995; Stone and Stoneking 1998). Therefore the mitochondria of each generation will be identical to the maternal population of their direct ascendants and descendants. These mitochondria accumulate mutations, or substitutions, at a much higher rate than the nuclear genome (Larcombe 2005). In each successive generation, as mutations are acquired, genetic distance will accumulate (Larcombe 2005), this permits researchers to trace lineages backwards and estimate chronological distance. A comparison of the hypervariable regions of two random individuals would reveal differences in 3 out of every 100 nucleotide positions (Stoneking 2000), indicating a non-relation, or a very distant relation. Closely related individuals, such as mother and child, should manifest no differences in the same comparison although mtDNA can also be used to identify individuals (Brown 1980).
For this research mtDNA was employed to ensure that the individual being analysed was a member of the target population. Elimination of individuals with mitochondrial profiles outside of expected haplogroups was essential to accurately assess and authenticate the results extracted from nuclear DNA analyses. A limited number of haplogroups are known to exist within ancestral groups of North, South and Central America allowing for simple exclusion based on haplogroup membership. Due to the fact that the Arctic has experienced only a short period of prolonged contact with populations outside of the Inuit ethnic group it is highly unlikely that a significant amount of gene flow had occurred within the population analysed. Individuals from the pre-contact period could be definitively assigned to the five known haplogroups of North America, and any individuals exhibiting a haplogroup outside this limited number are easily assigned the determination of contaminated and excluded from analysis.
Polymorphisms in the HVR regions of mtDNA can be used to trace descent populations and migration patterns (Hagelberg and Clegg 1993). Haplogroups, groups of individuals that share similar mitochondrial genetic markers but that are not necessarily closely related (Larcombe 2005), have been traced for the global population. To date five haplogroups, and a variety of haplotypes, have been assigned to New World aboriginal populations based on mitochondrial restriction/deletion polymorphisms: A, B, C, D, and X (Torroni et al. 1992), however haplogroups A-D demonstrate virtually all of North and South America's mtDNA diversity (Tamm et al. 2007). A2, B2, C1, D1 are the four major pan-American haplotypes and are thought to be the founding New World h