Published ahead of print on April 17, 2008, doi:10.1164/rccm.200710-1554OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200710-1554OC
Mapping of a Novel Susceptibility Locus Suggests a Role for MC3R and CTSZ in Human Tuberculosis 1 Wellcome Trust Centre for Human Genetics, University of Oxford, Churchill Hospital, Headington, Oxford, United Kingdom; 2 Imperial College, London, United Kingdom; 3 Africa Centre for Health and Population Studies, University of KwaZulu-Natal, Somkhele, South Africa; 4 London School of Hygiene and Tropical Medicine, London, United Kingdom; 5 National TB/Leprosy Control Programme, Banjul, The Gambia; 6 Medical Research Council Laboratories, Fajara, The Gambia; 7 CHU Ignace Deen, Conakry, Guinea; 8 Danish Epidemiology Science Centre, Bissau, Guinea-Bissau; 9 Karonga Prevention Study, Chilumba, Malawi; and 10 Medical Research Council Centre for Molecular and Cellular Biology, Stellenbosch University, South Africa Correspondence and requests for reprints should be addressed to Graham S. Cooke, Ph.D., M.R.C.P., Africa Centre for Health and Population Studies, PO Box 198, Mtubatuba 3935, Somkhele, KwaZulu-Natal, South Africa. E-mail: gcooke{at}africacentre.ac.za
Rationale: Tuberculosis remains a major cause of morbidity and mortality in the developing world. A better understanding of the mechanisms of disease protection could allow novel strategies to disease management and control. Objectives: To identify human genomic loci with evidence of linkage to tuberculosis susceptibility and, within these loci, to identify individual genes influencing tuberculosis susceptibility. Methods: Affected sibling pair analysis in South African and Malawian populations. Independent case-control study in West Africa. Measurements and Main Results: Two novel putative loci for tuberculosis susceptibility are identified: chromosome 6p21-q23 and chromosome 20q13.31—33—the latter with the strongest evidence for any locus reported to date in human tuberculosis (single point LOD score of 3.1, P = 10–4, with a maximum likelihood score [MLS] of 2.8). An independent, multistage genetic association study in West African populations mapped this latter region in detail, finding evidence that variation in the melanocortin 3 receptor (MC3R) and cathepsin Z (CTSZ) genes play a role in the pathogenesis of tuberculosis. Conclusions: These results demonstrate how a genomewide approach to the complex phenotype of human tuberculosis can identify novel targets for further research.
Key Words: tuberculosis • host genetics • MC3R • Africa
Interest in human susceptibility to tuberculosis predates the discovery of the causative pathogen. Evidence has accumulated from a number of sources that human genetics influences an individual's susceptibility to disease and in recent years some of the genes responsible have been identified, most notably HLA class II (1–3), natural resistance-associated macrophage protein-1 (NRAMP1) (4–8), interferon gamma (IFNG) (9–11), and MYD88 adaptor-like (MAL) (12). These successes have generally used a candidate gene approach based on knowledge of human immunology gained from bench, animal, and human studies. Genomewide linkage studies offer the promise of discovering genes influencing disease susceptibility for which a role might not have been previously suspected. To date, despite success in the closely related disease of leprosy (13–16), genomewide linkage studies have failed to identify a major susceptibility locus for tuberculosis. Previous work in a South American study (17) found the strongest evidence for linkage in the genomic regions 20p12.1 and 17q11–21, and work in African populations (18) has implicated the regions 15q11–13 (19) and Xq27 (20). We performed further affected sibling pair linkage analysis in two populations: "Coloureds" (South Africans of mixed racial origin) from metropolitan Cape Town and Malawians from the Karonga district. One genomic region, 20q13.31–33, was prioritized for further study. With a two-stage design, this region was then mapped in detail in a large, independent West African case-control study, with the aim of identifying novel loci involved in the pathogenesis of disease.
Family Recruitment Families for genomewide analysis were recruited from the Coloured population in metropolitan Cape Town, South Africa. A total of 131 sibling pairs were identified in 81 families with pulmonary tuberculosis, defined by either a positive sputum smear or positive mycobacterial culture. Twenty-four sibling pairs were identified in 24 families recruited from the Karonga Prevention Study in Malawi. Where possible, DNA was also collected from both parents. When this was not possible, DNA from unaffected siblings was collected to allow reconstruction of parental genotypes. All individuals gave informed consent for their samples to be used for genetic analysis. Approvals were obtained from local ethics committees (University of Oxford, UK; Gambian Government/Medical Research Council Ethical Committee; Ministry of Public Health, Guinea-Bissau; National Ethics Committee, Ministry of Health, Conakry, Guinea; and the ethics committee of the Faculty of Health Sciences, Stellenbosch University, South Africa).
Microsatellite Genotyping
Statistical Methods Maximum likelihood multipoint mapping was performed with the Mapmaker/Sibs program (Whitehead Institute for Biomedical Research, Cambridge, MA) (23) to increase the information content at any given locus. Only fully independent sibling pairs were analyzed and maximum likelihood scores calculated using Holman's constraints (24). Multipoint data were assessed for possible errors in genotyping using PEDwipe, part of the Merlin program (University of Michigan, Ann Arbor, MI) (25).
Analysis
Case-Control Study
SNP Typing
Genomewide Analysis Using an LOD score of less than or equal to –2 as a threshold for exclusion, over 40% of the genome was found unlikely to contain a putative disease susceptibility locus with a locus-specific  s of 2 or more. The first-round genome screen identified 64 markers that were followed up for further analysis. A novel region, not previously studied in this population, was identified on chromosome 6 (6p21-6q23) with evidence for linkage found in both stages of the genomewide screen. The highest single point of linkage was found at marker d6s434 (single point LOD, 1.9; MLS, 1.82; P = 0.002). Evidence for linkage was found within the region of 15q11-13, identified in previous work (18), and including samples previously analyzed, an MLS (LOD) score of 2.1 is found in the Coloured population alone. Further evidence suggesting linkage was identified in regions including 20q31-33 (LOD score on first round, 0.9; overall MLS, 0.6) and 14q31-32 (LOD, 1.1 on first screen; overall MLS, 0.9). There was weak evidence for linkage in the region 17p11.2-17q11.2 (MLS, 0.6). Twelve markers used in the complete genome screen were then selected for study in the Malawian population. These 12 markers were drawn from the LMSv2 and chosen if they fell within the regions of suggestive linkage on chromosomes 6p21-q23, 15q11-13, 20q13.31—33, and Xq27. A list of these markers is available from the authors. Two of these markers were within the putative region of linkage on chromosome 20 (d20s171 and d20s173); d20s171 showed evidence of linkage (LOD, 2.0; P = 0.0005) with some weak evidence for d20s173 (LOD, 0.91; P = 0.91). To prioritize regions for further study, all markers typed with the same primers were analyzed together with families studied previously (18) as a single dataset. The strongest evidence for linkage was within the region 20q13.31-33 (with an MLS of 2.8 on multipoint mapping, P = 0.00008; see Figure 1).
Detailed Association Mapping of 20q30.31-33 In an initial screen of 40 SNPs, polymorphism within two genes showed evidence of disease association in the Gambia: melanocortin 3 receptor (MC3R; rs3827103 referred to here as MC3R241) encoding MC3R (MC3R241 genotype AA protective, P = 0.04) and CTSZ (GenBank SNP NM_011362 [refSNIP rs34069356; National Center for Biotechnology Information, Bethesda, MD] referred to here as CTSZ3P) encoding cathepsin Z (CTSZ3P genotype CC susceptible, P = 0.01). Associated polymorphisms within these genes were typed in the populations of Guinea-Bissau and Guinea.
CTSZ Genotyping was repeated to confirm the results and 90 cases and control subjects were sequenced to validate the genotypes. Once strong disease association was confirmed within the 3'UTR SNP, sequencing was extended to the gene's coding exons and other neighboring markers were examined in more detail. Only one nonsynonymous SNP was identified within CTSZ (in exon 5), but this showed no evidence of disease association (P = 0.31).
MC3R In light of the association seen in the Gambian population within TBGENENV, further genotyping was done in samples from a previous study (4). This independent dataset also found evidence of a protective role for the MC3R241 AA genotype. Genotypes found in cases were AA 63 (22.2%), AG 137 (48.2%), and GG 84 (29.6%), and in control subjects were AA 53 (30.9%), AG 68 (39.7%), and GG 50 (29.2%); OR for the AA genotype, 0.63; 95% CI, 0.40–1.00; P = 0.04. Combining the two Gambian populations found a stronger protective effect (OR, 0.67; 95% CI, 0.50–0.88; P = 0.004).
There are two key aspects to these results: the first is the novel identification of a major locus for susceptibility to human pulmonary tuberculosis on chromosome 20; the second is the identification of two susceptibility genes with a plausible link to tuberculosis, both of which merit further study. The great attraction of linkage analysis remains that, starting from clinical cases of disease, and with no prior hypothesis of disease immunology, it is possible to identify previously unsuspected molecules playing a role in disease. For tuberculosis, there are three potential susceptibility loci that have been studied in detail to date. Those on 15q11-13 and Xq27 (18) have the most statistically convincing evidence of linkage in published work to date. The former has already been the subject of some preliminary mapping (19) with the gene UBE3A implicated in disease. Key candidates within Xq27 have also been investigated and no disease association was established for either of two candidate genes prioritized on the basis of their position and function, CD40 ligand, and iduronate 2-sulphatase (IDS) (20). More recently, detailed mapping within a locus on chromosome 17q11-q21 has found disease association with several genetic variants within one gene cluster (28), which could not be resolved more finely. The data presented here suggest two novel putative tuberculosis susceptibility loci in African populations, 6p21-23 and 20q13.31-33. The latter presents the strongest evidence for linkage yet with the complex phenotype of tuberculosis. The challenge of finding disease association beyond the original dataset can often be difficult and confounded by the problems of multiple testing. The multistage strategy described here finds evidence of disease association within the gene encoding cathepsin Z, a member of the cathepsin protease family, and MC3R, a member of the melanocortin receptor (MCR) family. The cathepsins are a family of cysteine proteases, which form a major component of the lysosomal proteolytic system and thus potentially have a role in immunity to pathogens including Mycobacterium tuberculosis. Endosomal proteases have a role in two elements of major histocompatibility class II–restricted antigen presentation: antigen degradation and invariant chain cleavage. The gene encoding cathepsin Z was cloned after a search for homologs of the then-known cathepsins (29). The enzyme, 303 amino acids in length, has significant similarity to cathepsin B (26%), cathepsin C (34%), and cathepsin L (30). However, the observations that the enzyme differs significantly from other cathepsins with a shorter leader propeptide, a more diverse array of insertions around the active site, and an unusual chromosomal location led the authors to suggest it might be a member of an independent cathepsin subfamily. It is encoded by a gene within the region 20q13.31-33. To date, evidence supporting a role for cathepsin Z in tuberculosis has been inferred from an appreciation of its role in macrophages and by analogy to other cathepsins. Cathepsin Z is one of only two cathepsins preferentially expressed in murine macrophages (the other is cathepsin F) (31). It is recruited to the murine phagosome and is found there hours after phagocytosis (32). Using murine primary macrophage cell lines (J774), Lennon-Dumenil and colleagues monitored the sequential recruitment and activation of cathepsins to the phagosome. They found that cathepsin Z was one of the four most abundant cysteine proteases present and active in early phagosomal compartments, despite its relatively mild pH, in contrast to other cathepsins that were recruited later (33). That this pattern of recruitment and activation was not observed in a dendritic cell line suggests that the role of cathepsin Z might be specialized to macrophages and, by analogy to other cathepsins, host defense. In contrast to a role in protective immunity, cathepsin Z could mediate tissue damage by degradation of the extracellular matrix. For example, it has been reported that cathepsin Z is strongly overexpressed in malignant melanoma cells and could play a role in allowing tumors to metastasize. MC3R is part of a wider family of MCRs that have been of key interest in the study of obesity and weight control. They respond to natural melanocortin proteins derived from the larger molecule POMC (pro-opiomelanocortin), and share a single amino acid motif (HFRW sequence). The MC3R protein is 361 amino acids long and encoded by a single exon on 20q13.2. The molecule is a G-protein–coupled membrane receptor with seven transmembrane domains.
The different receptors within the MCR family differ in their specificity for particular POMC derivatives (34), their tissue expression, and their biological effects once activated. MC3R differs from the other melanocortins in that it has no selective specificity for melanocortin-derived products and is the only MCR strongly activated by
Beyond energy homeostasis, it has been realized for some time that derivatives of POMC have antiinflammatory effects in a variety of animal models of inflammation, including mycobacterial arthritis (35, 36). MC1R was originally believed to be the key family member playing a role in inflammatory responses, mediating the proinflammatory effects of The size of the effects seen for both CTSZ and MC3R means it is very unlikely that variation in either gene plays a major role in tuberculosis, at least in the populations studied. However, these results are the first direct suggestion of a role for members of either the cathepsin family or the MCRs in the pathogenesis of tuberculosis, a hypothesis that can now be tested in genetic, cellular, and clinical studies, and that offers potentially important insights into the pathogenesis of this persistent disease.
The authors thank all the participants in this study. Sadly, since this study was conceived, Professor Steve Bennett and Frank Mwangulu have passed away. The authors also thank Lifted Sichali, David Warndorff, Mia Crampin, and Steve Chaguluka.
Supported by the Wellcome Trust and the EC (contract no. IC18CT980375). Sample collection in the Gambia was supported in part by the Medical Research Council (no. G0000690 to G.S.).
Originally Published in Press as DOI: 10.1164/rccm.200710-1554OC on April 17, 2008 Conflict of Interest Statement: G.S.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.J.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.B. is deceased and is unable to provide a conflict of interest statement. C.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.P.W.J.M. has been working in Uganda for 3 years at the Infectious Diseases Institute where Pfizer, Inc., Gilead, Exxon Mobil, and Becton Dickinson have been donors. None of these donors supplied work in the Gambia, where they did not have commercial sponsorship. G.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. O.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.M. is deceased and is unable to provide a conflict of interest statement. P.v.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.G.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.V.S.H. is a nonexecutive shareholding director in Oxxon Therapeutics PLC. Received in original form November 22, 2007; accepted in final form April 11, 2008
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