Human genetic susceptibility to mycobacterium tuberculosis 1

Human Genetic Susceptibility to M. tuberculosis

Credit seminar 2nd semester

Submitted by:Shweta kaul

Master of life sciences with specialization in Human GeneticsRegistration number:15mslshg02

Centre of Human Genetics and Molecular MedicineSchool of Health Sciences

Central university of Punjab, Bathinda, 151001

1/29/2016 1

Tuberculosis It is a contagious disease caused by the airborne bacterium M.

tuberculosis Infects primarily lungs causing primary infection Pulmonary TB and Extra pulmonary TB Spreads through lymph nodes and bloodstream to any organ of

the body Contagious as can spread through cough, sneeze, or speak Person suffers cough, weight loss, loss of appetite, fever,

coughing up blood and night sweats Difference between active form of disease and infection of lungs Touching someone who has disease does not necessarily spread

the disease

1/29/2016 Central university of Punjab: HGMM 2

Tuberculosis

• Two stages – pulmonary TB and extra pulmonary TB(in very rare cases example in immunosuppressed individuals)

Pulmonary TB

Activation of second line of defence

Formation of granuloma

Caused by bacilli M. tuberculosis

Infection of lungs

Dormant form

Macrophage inflammation

reactivation

3Central university of Punjab: HGMM1/29/2016

History and evidences of TB susceptibility

• Disease first identified by Robert Koch in 1882• In 1929, Lubeck , Germany a complete lot of

BCG vaccine was contaminated with M. tuberculosis

• Vaccine injected in 251 infants• 4 remained uninfected, 72 died within a year,

175 overcame• 70% of naive babies survived the infection

Reference: Moller % Hoal; 2010


Reference: Bellamy, 1998

1/29/2016 5Central university of Punjab: HGMM

Epidemiology of the disease

• 1/3rd of world population is affected with this infection

• But only 1/10th of this develops the active form of the disease

• 10 million of US population is infected with this infection

• Amongst top 3 causes of death in India and 2/3rd of all the cases in India are from underprivileged groups and out of this 36% new cases are from immigrants


Reference: (Moller andHoal, 2010; Swaminathanand Rekha, 2010)


Figure 3(a). Pie chart showing the incidences of TB in the year 2012 worldwide (Source: Global Tuberculosis report, 2013)

Figure 1. Incidence rate map of the disease in the year 2012 worldwide (Source: www.cdc.gov; 2012)

http://www.cdc.gov/


Figure 2. Pie chart showing the incidences of TB in the year 2012 worldwide

(Source: Global Tuberculosis report, 2013)

Interaction between human and M. tuberculosis

Pathogen Associated Molecular Patterns interacting with Pattern Recognition Receptors' for example Toll Like Receptors and Non- Toll like receptors (mannose binding receptors, lipopolysaccharide

binding receptors)

Cascade of reactions get activated

Cytokines example Tnfα/ IL 12/ IL 1 are secreted

These cytokines activate second line of defence

Phagocytosis/ apoptosis/ opsonisation/ inflammation

Source:www.docmeg.wordpress.com,2011Central university of Punjab: HGMM1/29/2016

Source: www.newint.org,2013

www.cdc.org, 2004

http://www.cdc.org/

Central university of Punjab: HGMM 10

Growth of bacterium inside the host

• The bacterium is unique in having lipid cell wall composition more than peptidoglycan

• Mycolic acid (alpha branched lipids)• Cord factors (trehalose mycolate)• Wax-D (peptido-glycolipid)

1/29/2016

Reference: Meena ET al., 2012

Survival mechanism adapted by M. tuberculosis


Figure3. Key survival mechanisms involved in phagosome maturation arrest ofM. tuberculosis inside the macrophages

(Source: Meena et al., 2012)

Some of the Pathogen genes interacting with host

Gene Function

Erp gene Encodes exported repetitive proteins, these surface proteins

function in virulence

Mas gene Marker assisted selection, encodes mycoserosic acid synthase

that catalyses the synthesis of mycoserosic acids that helps

in protection against macrophages

Fbp A Fibronectin binding protein A, encodes mycolyl transferase

enzyme that helps in growth of bacteria

Omp A Outer membrane protein A precursor, porin like proteins

forms pores in liposomes of host

Hbh A Encodes Heparin binding hemagglutinin protein , binds heparin

and

promotes hemagglutination, this helps in invading the host

defence mechanism

Lam Laminin encoding genes, laminin depress IFNγ production

Host susceptibility mechanisms

• Upon infection there are chances that the host can either develop or resist the disease

• Based on expression of resistance and susceptibility genes

• These genes majorly grouped into two types HLA and Non HLA genes


This association can be well understood by below three hypothesis

• Hypothesis I

Hypothesis I

HLA Antigen is getting influenced by the bacterium

M. tuberculosis and doing its molecular

mimicry.

HLA antigens act as receptors for the

bacterium M. tuberculosis.

HLA antigens are involved in

weakening the immune responses.

reference: selvaraj, 2004


Hypothesis II :MHC genes are present in closer proximity to

genes that are involved in susceptibility.

Hypothesis III:Non-HLA genes are involved.

reference: Selvaraj, 2004

1/29/2016 15Central university of Punjab: HGMM

Classification of susceptibility genes


(A).HLA and Non HLA gene

type

(B). Genes involved in

various immunological

processes

(A). HLA and Non HLA gene HLA genes• An increased antigen

recognition range by different polymorphic forms is seen

• to antigen specificity• Susceptibility allele example:

HLADRB1 to HLADRB14• In Chinese and Kazak

population: HLADQB1 and HLADRB1 genetic polymorphisms reference:Khalilullah

et al., 2014)

Non HLA genes • Genes other than HLA are

responsible for susceptibility• Haptoglobulin genes

polymorphism • Three polymorphic forms of

genes:HP1-1; HP2-1; HP2-2• HP1-1 is most

effective ;HP2-1 is moderately active while HP2-2 is Least active


(B). Genes involved immunological processes


Transporter genes

Receptor encoding

genes

Genes involved

in bacterial killing

Genes involved

in apoptosis

of I CChemoki

ne encoding

genes

Cytokine encoding

genes

Genes involved in A P

Genes involved in OP of M. tuberculosis

Reference: Moller and Hoal, 2010


Figure4. Various genes involved in susceptibility mechanism (source: Moller and Hoal, 2010)

Genes involved in antigen presentation

Type Human leukocyte antigenGene name HLA DR, HLA DQ, HLA DP, HLA DM, HLA

DOA, HLA DOBpresent on MHCII

HLA A, HLA B and HLA C present on MHCIChromosomal location Chromosome 6 , both short and long armsFunction - Encodes cell surface antigen presenting proteins

- MHC I , HLA genes present antigens to CD8+, Cytotoxic T cells and present antigens from inside the cell

- MHC II, HLA genes present antigens to CD4+, helper T cells and present antigens from outside the cell

Effect Increased expression of the mutant form or recessive polymorphic form of allele results in higher susceptibility levels or less expression of wild type allele leads to susceptibility

Polymorphic form/forms HLA DR2 Subtype -DRB1*1501*1502 -DRB1*0601HLA DQ1 -DQB1*0601HLA DP -DPB1*02Haplotype -DRB1 and 1501-DQB1*0601 -DRB1*11, DBB1*10, DQB1*0501

Epidemiological studies Except Haplotype –DRB1*11, DRB1*10, DQB1*0501 leads to resistance, all other polymorphisms are responsible for susceptibility amongst Indian population studies

References (Kindt et al., 2007 ; Moller and Hoal, 2010; Balamurugan et al.,2004)


Type Non classical HLA

Gene name Tap2 and Tap1

Chromosomal location 6q;(between HLA DQ and HLA DP)

Function - These genes encode transporter associated proteins- These proteins present antigens to CD8+, Cytotoxic T cells i.e. help in

cellular mediated immune responses- The polymorphic residues of these genes provide specificity in antigen

presentation


Polymorphic form/forms Tap1 -rs1135216A/G

Tap2 -rs241447

Epidemiological studies -rs241447 Decrease risk of TB in Iranian population studies

-rs1135216A/G In Iranian population homozygote GG and heterozygote AG increased the risk of TB as compared to homozygote AA

References (Kindt et al.,2007 ; Naderi et al., 2015)

Cytokine encoding genes


Type Non-HLA genes

Gene name IFNG

Chromosomal location 12q and 10q

Function - It encodes cytokine Interferon-γ , that activates macrophages and is produced by T-cells - It also helps in antigen presentation to CD8+, Cytotoxic T cells


Polymorphic form/forms

IFNG+874(A/T) polymorphism of Intron regionHaplotype- TA, TC, CC

Epidemiological studies - +874(A/T) polymorphism is seen to be responsible for susceptibility in Pakistan, China, Hong Kong, Sicilian, South Africa; but no association of this SNP is seen in Malawian, Houston, West Africa, South Indian and Croatian population

- +874 A is responsible for susceptibility while +874 T is responsible for protection against TB- Polymorphism -56(CC) in promoter and -56(CA) in Intron region are responsible for

susceptibility - In case of Haplotype – over expression of TA in North Indians is responsible for

susceptibility

References (Kindt et al., 2007; Abhimanyu et al., 2012; Moller andHoal, 2010;

Khalilullah et al., 2014)

Type Non-HLA genesGene name IL12B, IL1B, IL1RA, IL6, IL10Chromosomal location IL12B 5q

IL1B 2qIL1RA 2qIL6 7pIL10 1q

Function IL12B - Encodes cytokine Interleukin12-β that is produced by activated macrophages, monocytes, B lymphocytes and dendritic cells

- This molecule helps in inducing, maintaining the number and memory of Helper T cells

IL1B - It encodes cytokine Interleukin1β that is a pro-inflammatory molecule and is produced by monocytes, macrophages, dendritic cells

- Its level increases during defence mechanism

IL1RA - It encodes cytokines IL1μ and IL1β - It helps in inhibition of IL1 activity on T cells, therefore act as anti-inflammatory cytokine

IL6 - It is expressed in hepatocytes, monocytes, B cell lymphocytes, macrophages and neutrophils as a anti inflammatory cytokine

- Expression blocks the IFNγ mediated signalling- It is also involved in macrophage and cytotoxic T cell differentiation it also induces IL-4 production- Higher levels of IL-6 are involved in resistance

IL10 - It expressed cytokine has anti-inflammatory properties- The cytokine acts as an antagonist (i.e. opposes) Tnf-α, Ifn-γ and IL-12B production as they are the cytokines

that lead to inflammation- It is produced after the M. tuberculosis bacterium has been phagocytosed via monocytes, macrophages and

T-lymphocytes

Effect The over expression of IL6, IL1RA and IL10 leads to susceptibility while others are responsible for susceptibility in cases of less expression


IL10 -1082(G/A) and -592(A/C)(both in promoter region)-2849A, -819C

IL6 -572C>G, -572(GG)Epidemiological studies -1082(G/A) and -592(A/C)(both in

promoter region)-1082A is seen to be responsible for susceptibility in Ghana

-2849A, -819C -2849A is susceptible in Ghana-572C>T, -572(GG) In Ugandan population SNP-572C>T leads to resistance against TB while SNP-

572GG protects TB by IL-6 productionReferences (Kindt et al., 2007; Abhimanyu et al., 2012; Santos et al., 2002;

Acton et al., 2013; Khalilullah et al., 2014)

Type Non HLA genes

Gene name TNFA

Chromosomal location

6p

Function - It encodes cytokine Tumor necrosis factor-α , which is produced mainly by monocytes and macrophages

- Tnfα also signals these cells to enter the pulmonary system- It promotes the granuloma formation i.e. inactive form of TB



−1031 (T/C), −863 (C/A), −857 (C/T), −308 (G/A), −238 (G/A), −1196 (C/T), −1125 (G/C), −572 (A/C), −316 (G/A), −163 (G/A), and −70 (G/A)

Epidemiological studies

- -238(G/A) polymorphism is common in many countries- -238(G/G) polymorphism leads to resistance for the disease- -857(C/T) polymorphism is responsible for increase in susceptibility

as seen in Iran linkage studies References (Selvaraj, 2004; Santos et al., 2002; Abhimanyu et al., 2012;

Khalilullah et al., 2014)


Chemokine encoding genesType Non-HLA genes

Gene name CCL2

Chromosomal location

17q

Function The expression of this gene leads to the formation of monocyte chemoattractant proteins(MCP)



-2518(A/G) in promoter region


- -2518(A/G) has been studied as candidate susceptibility gene in Ghana- -2518A is associated with low MCP production leading to susceptibility for TB

as seen in Mexican, Korean, Chinese and Peruvian population studies- -2518G is associated with high MCP production leading to resistance - No association of this polymorphism was found with TB in South Indians,

Coloreds and Ghanians

References (Raja,2004; Khalilullah et al., 2014)


Metal ion transporter associated genes


NRAMP1expression

Bactericidal effects

Antiports iron bivalent cation

This cation localizes to the membrane of late endosomes or lysosomes

This makes the phagosomal environment acidic

Killing M. tuberculosis

Type Non-HLA genes

Gene name NRAMP1/ SLC11A1

Chromosomal location 2q

Function - It encodes Natural resistance associated macrophage protein which acts as antiporter or transporter

- This protein is expressed inside the macrophages which has bactericidal effects



3’UTR, D543N, 5’(GT)n and INT4-rs3731865, -rs17221959, -rs3731863


INT4 and D543N - Responsible for more TB susceptibility in Chinese population

- In Indonasian population , no association seen between D543N, INT4 and TB susceptibility

-rs3731865, -rs17221959, -rs3731863 - Responsible for susceptibility in US(Caucasians and African Americans)

References ( Moller and Hoal, 2010; Kozakiewicz, et.al., 2013; Khalilullah et al., 2014)


Receptor encoding genes Type Non-HLA genesGene name CD209Chromosomal location

19p

Function - These encode the surface molecules that provide the targets for antigen presentation on T cells

- These surface molecules are transmembrane protein predominantly expressed on dendritic cells, whereas its presence on macrophages depends on the tissue type and state of activation.

- It is induced in alveolar macrophages from M. tuberculosis infected patients. It directly mediates phagocytosis of M. tuberculosis by dendritic cells.



-871


This polymorphic form has been studied as candidate susceptibility gene in South Africa

References (Khalilullah et al., 2014; Moller and Hoal ,2010)


Type Non-HLA genes

Gene name VDR


Function - Enhances phagosomal activity of the cell


Polymorphic form/forms Variants Taq1(TT, Tt, tt), Apal(AA,Aa,aa), Bsml(BB, Bb, bb) and Fokl(FF, Ff, ff)

Epidemiological studies Fokl(FF, Ff, ff) - In Indonasian, Asian and Chinese population (FF) Fokl variant was expressed more in TB patients while levels of other variants didnot more differ within the groups

Bsml(BB, Bb, bb) - In Turkey, Bsml(BB) is associted more with TB susceptibility

Apal(AA,Aa,aa) - Apal in West African candidate gene susceptibility studies

Taq1(TT, Tt, tt) - No association was seen for VDR polymorphism in African and American population

References (Zhabagin et al., 2013 ; Moller and Hoal, 2010; Khalilullah et al., 2014;

Gao et al, 2010; Bornman et al., 2004)


Central university of Punjab: HGMM 31

Type Non-HLA genes

Gene name TLR2TLR9

Chromosomal location TLR2 4qTLR9 3p

Function These encode proteins that binds to PAMP’s


Polymorphic form/forms TLR2 R753Q(exon),R677(exon), -597(T/C) and P631HInsertion/ Deletion (-196 to -174)

TLR9 -rs352143Epidemiological studies R753Q(exon),R677(exon), -597(T/C), -

975(C/T) and P631H- -597(T/C) responsible for more

susceptibility for the disease in children

- -P631H expressed more in Croatian Caucasian TB patients

- -975(C/T) polymorphism is responsible for resistance in against TB in Korean population studies

Insertion/ Deletion (-196 to -174) Susceptibility in Guinea-Bissau and US (Caucasians) population for this polymorphism is seen

-rs352143 Susceptibility in US(African American) population for this polymorphism is reported

References (Khalilullah et al., 2014; Moller and Hoal, 2010)

1/29/2016

Genes involved in bacterial killingType Non-HLA genes

Gene name NOS2A/ iNOS


Function Enzyme encoded by this gene is present in cytosol(nitric oxide synthase)This enzyme catalyzes the production of nitric oxide


Polymorphic form/forms -CCTTT-TAAA-rs9282799, -rs8078340-rs2274894

Epidemiological studies -CCTTT High frequency in TB patients in North Western and Colombian population

-TAAA No association was seen with this polymorphism

-rs9282799, -rs8078340 Candidate susceptibility gene studied in South Africa

-rs2274894 Candidate susceptibility gene studied in US( African American)

References (Khalilullah et al., 2014; Moller and Hoal, 2010)


Genes involved in apoptosis of infected macrophage

Type Non-HLA gene

Gene name SP110


Function - It encodes speckled proteins that are nuclear body proteins- These proteins are responsible for apoptosis of infected macrophages- This process is induced by IFNG signals



-rs3948464-rs2114592


-rs3948464 -susceptibility in Ghambian population

-rs2114592 -susceptibility in republic of Guinea and Gambia

References (Khalilullah et al., 2014; Moller and Hoal, 2010 )


Genes involved in opsonisation of M. tuberculosis

Type Non-HLA genes

Gene name MBL


Function - It encodes mannose binding protein(secreted by liver), which binds to mannose and N-acetylglucosamine

- This is further followed by lysis of the cell wall of M. tuberculosis by MAC formation

- If MBL is in lesser amounts, the macrophage interacts with the bacilli directly- This interaction leads to opsonisation and phagocytosis

Effect Lesser expression of this gene leads to resistance for the host due to direct opsonisation by macrophages(i.e. without going to complement pathway)

Polymorphic form/forms Allele B Mutation in codon 54Allele C Mutation in codon 57Allele D Mutation in codon 52

Epidemiological studies These variants are associated with resistance to TB in Ghana

References (Kindt et al., 2007;Acton, 2013; Kozakiewicz et al., 2013;

Khalilullah et al., 2014; Soborg et al., 2003; Moller and Hoal, 2010 )



(Source: Soborg et al., 2003)

Figure5. Interaction of MBL and pathogen

A. B.

C.

Applications of studying the susceptibility genes

Identification of susceptible genes will help in:

Disease treatment:

Identification will put forward the susceptibility genes responsible

Protein responsible is identified

Biochemical pathway, Cytokines, and all the steps can be studied

Gene targeting can be done

37Central university of Punjab: HGMM

1/29/2016

Genetic testing:

Development of new vaccines: These studies has revealed majorly the involvement of Non-HLA genes

• Development of new vaccines as the targets become more vast

• But this application is still in its infancy

By identification of the gene responsible population at risk can be identified

Genetic therapy can be processed

Most possible application


Prevention, Diagnosis and Treatment of Tuberculosis

Prevention of TB• BCG(Bacillus of Calmette and Guerin) vaccine:

named after two Frenchmen who developed it• It consists of live attenuated strain(virulent part

removed) of M. bovis (avirulent)• Drawbacks: TB reactivation cannot be prevented It prevents the disease formation but not the infection Complicates the way TST operates banned in U.S.


Diagnostics of TB• Ziehl neelson method:

Sputum(coughed up saliva) taken

Treated with NaOH(except M. tuberculosis other pathogens get killed)

Cultured on BACTEC media (Becton Dickinson) , it contains radiolabelled palmitate as a sole carbon source

As the bacterium divides , palmitate divides liberating radiolabelled carbon dioxide

Which detects growth within 9-16 days


Mantoux test/ Tuberculin skin test/ PPD(purified protein derivative)


• Treatment of TB: Common TB drugs are:

• Moxifloxacin is a fluroquinolone antibiotic drug that targets DNA gyrase and kills bacteria by interfering with its replication process. This drug showed huge potency against TB than other TB drugs. This drug can shorter the treatment from 6 months to 3-4 months

Drug Mode of action

Rifampicin inhibits bacterial RNA polymerase

Isoniazid inhibits the synthesis of mycolic acid

Ethambutol obstructs the formation of cell wall

Streptomycin acts as a protein synthesis inhibitor


Conclusion

• Various number of susceptibility genes such as HLA genes and Non-HLA genes and their polymorphic forms responsible for the Tuberculosis has been studied

• This gives a vast area of study for treatment by targeting these genes

• But many genes are yet to be identified• The challenges for study and treatment are yet to

overcome due to co evolution of the bacterium and human both

Central university of Punjab: HGMM 431/29/2016

Future perspectives

• Challenges in TB susceptibility studies - GWAS• Human and M. tuberculosis co-evolution• Schistosoma mansoni co-infection

increased M. tuberculosis susceptibility• MicroRNA-223 in TB susceptibility


References • Abhimanyu, M. B., Jha, P., and Indian Genome Variation Consortium. (2012). Footprints of

genetic susceptibility to pulmonary tuberculosis: Cytokine gene variants in north Indians. The Indian journal of medical research, 135(5), 763.

• Bellamy, R. (1998). Genetic susceptibility to tuberculosis in human populations. Thorax, 53(7), 588-593.

• Bornman, L., Campbell, S. J., Fielding, K., Bah, B., Sillah, J., Gustafson, and Sylla, A. (2004). Vitamin D receptor polymorphisms and susceptibility to tuberculosis in West Africa: a case-control and family study.Journal of Infectious Diseases, 190(9), 1631-1641.

• Dorhoi, A., Iannaccone, M., Farinacci, M., Faé, K. C., Schreiber, J., Moura-Alves, and Heinemann, E. (2015). MicroRNA-223 controls susceptibility to tuberculosis by regulating lung neutrophil recruitment. The Journal of clinical investigation, 123(11), 4836.

• Dunn, P. L., and North, R. J. (1995). Virulence ranking of some Mycobacterium tuberculosis and Mycobacterium bovis strains according to their ability to multiply in the lungs, induce lung pathology, and cause mortality in mice.Infection and immunity, 63(9), 3428-3437.

• Gao, L., Tao, Y., Zhang, L., and Jin, Q. (2010). Vitamin D receptor genetic polymorphisms and tuberculosis: updated systematic review and meta-analysis. The international journal of tuberculosis and lung disease, 14(1), 15-23.

Central university of Punjab: HGMM 451/29/2016

• Khalilullah, S. A., Harapan, H., Hasan, N. A., Winardi, W., Ichsan, I., and Mulyadi, M. (2014). Host genome polymorphisms and tuberculosis infection: What we have to say?. Egyptian Journal of Chest Diseases and Tuberculosis, 63(1), 173-185.

• Meena, L. S. (2010). Survival mechanisms of pathogenic Mycobacterium tuberculosis H37Rv. FEBS Journal, 277(11), 2416-2427.

• Moller, M., and Hoal, E. G. (2010). Current findings, challenges and novel approaches in human genetic susceptibility to tuberculosis. Tuberculosis,90(2), 71-83.

• Monin, L., Griffiths, K. L., Lam, W. Y., Gopal, R., Kang, D. D., Ahmed and Kolls, J. K. (2015). Helminth-induced arginase-1 exacerbates lung inflammation and disease severity in tuberculosis. The Journal of clinical investigation, 125(12).

• Raja, A. (2004). Immunology of tuberculosis. Indian Journal of Medical Research, 120(Oct), 213-232.

• Santos, A. R., Suffys, P. N., Vanderborght, P. R., Moraes, M. O., Vieira, L. M., Cabello, P. H. and Sampaio, E. P. (2002). Role of tumor necrosis factor–α and interleukin-10 promoter gene polymorphisms in leprosy. Journal of Infectious Diseases, 186(11), 1687-1691.

• Selvaraj, (2004). Host genetics and tuberculosis susceptibility. Current Science, 86(1), 115-121.

• Soborg, C., Madsen, H. O., Andersen, Å. B., Lillebaek, T., Kok-Jensen, A., and Garred (2003). Mannose-binding lectin polymorphisms in clinical tuberculosis. Journal of Infectious Diseases, 188(5), 777-782.

• Swaminathan and S.Rekha (2010). Pediatric tuberculosis: global overview and challenges. Clinical Infectious Diseases, 50(3), 184-194.

• Zhabagin, M., Abilova, Z., Askapuli, A., Rakhimova, S., Kairov, U., Berikkhanova, and Akilzhanova, (2014). Vitamin D Receptor Gene Polymorphisms in Susceptibility to Tuberculosis in the Kazakh Population in Almaty and Almaty Area. Central Asian Journal of Global Health,66(2).1/29/2016 Central university of Punjab: HGMM 46


`

Thanking you

Human genetic susceptibility to mycobacterium tuberculosis 1

Science

Transcript of Human genetic susceptibility to mycobacterium tuberculosis 1