Association Between Leptin Receptor Gln223Arg Polymorphism And Pulmonary Tuberculosis Relapse In Nigerian Patients.

Iwalokun Bamidele, A Presented at

Seventh EDCTP Forum 30 June – 02 July, 2014

Berlin, Germany

Overview •  INTRODUCTION

–  Global overview of TB burden –  Constraint to TB eradication with focus on TB relapse –  The Nigerian context : TB epidemiology and relapse –  Specific Objectives

–  METHODS –  RESULTS –  DISCUSSION –  FUTURE WORK –  ACKNOWLEDGMENT

Tb Burden Global Perspective, 2013

• 8.6M incidence (8.3 – 9.0)

• 12M prevalence (11 -13)

• 80% of cases concentrated in 22 countries

• Africa : 27% of cases

• 0.45M MDR-TB incidence (0.30 -0.60)

• XDR reported in 99 countries

INTRODUCTION

There has been progress towards TB eradication but at a slower rate globally.

New TB cases declining at 2% per year globally (20% decline rate needed for eradication) TB mortality reduced by 45% since 1990 (Target of 50% within reach) Increased access to Category 1 and Category II treatment regimens.

Duration/Cat I Phase Drugs combination

2 mo Intensive HERZ 5 mo HR

Duration/Cat II Phase Drugs combination

2 mo Intensive HERZ + S 1 mo HERZ 5 mo HER

First Line Retreatment

Constraints to TB Eradication : Numerous with new entrants

2billion per year funding gap (Diagnostics + treatment: Sus & MDR + RDTs + Lab strengthening + HIV/TB activities) ~ 3M missed cases (undiagnosed or un-notified) – Socioeconomic + health system factor MDR-TB prevalence with 27 HBC including Nigeria (84,000 MDR treatment eligible patients reported in 2012) MDR-TB treatment gap (18% globally but ~ 50% in Africa)

TB Relapse - Recurrence of active TB after successful treatment or completed treatment

Reportage of TB Relapse is on the Increase especially in low and middle income countries.

Country Study Design

Size

Determinant Intervention

Outcome/Authors

Morocco MDR rate (relapse) = 12.6%

Retros 232 •  Illicit drug use •  Low rate sputum

Smear conversion by 3 mo

•  Missed doses during intensive phase

•  Hospitalization

Cat II 74% success rate/Dooley et al, 2011

Brazil Relapse: 37 (4.9%)

Pros. Longitudinal

754 •  Smoking •  Lack access to

FHP •  Delay of 60 days to

start of treatment

- Batista et al, 2008

Brazil, 16% Relapse Rate

•  Missed consultation •  Irrational drug use •  Adverse events •  Irregular drug

supply

- Oliveira et al, 2000

Possible Mechanisms of TB relapse have yielded conflicting results”

Hypothesis: 1.  Morphological and functional changes in alveoli

macrophages (Mauya et al, 2002; Davies et al, 2006).

2.  Persistence of MTB in the lungs

3.  Reactivation of latent TB to active TB Studies with conflicting results Oliveira et al, 2000 Thomas et al, 2006

Mycobacterium tuberculosis and its host cell, the macrophage. 2000. Banky et al, 2000 . http://www.sp.uconn.edu/~terry/Spring96/WebTB2/Groups/Group5/

TB Relapse: Consequences vs. Determinants: need for identification of host genetic factors.

TB Relapse

Socio-economic factors:

Poverty

Low Education

Biologgical factors:

Age

Gender

Lifestyle factors

Alcohol

Smoking

Clino-epidemiological:

HIV

Delay initiation of ART

High viremia

Low CD4 count

MDR TB 10 infection

TB contact

Anaemia etc

Body weight gain

Leptin-LEPR polymophism not known-???

TB Relapse: Consequences

1.  Expansion of MDR-TB reservoir in the population

2.  Decreased category II success rate.

3.  Increased risk of XDR-TB

4.  Increased public health cost of treatment.

Determinants

Leptin: A Biomarker associated with TB development and severity???

TB with reduced serum leptin results in wasting (Crevel et al, 2002

Leptin increases during TB and further during treatment. Cakir et al, 1999

Leptin is associated with weight reduction and TB patients may not gain weight as expected despite successful treatments (Miller et al, 2000

X

Leptin’s biological activities are mediated by Leptin receptor 4 of the 6 isoforms in humans; isoform b the longest and most functional LEPR Gln223Arg influencing Leptin signalling (Laura et al, 2004; Pena et al, 2014)

Leptin signalling via the JAKS/STAT3 Pathway Cavva and Materese, 2004. Reviews Immunology, 4 (5), 371-379

Leptin (16 kDa protein from ob gene, chr 7) acts biologically through Leptin Receptor [1165 aa protein from 8.2 kb db gene (chr 1, 20 exons)]

FIG. Leptin and Metabolism Proccini et al, 2012Molecular aspects of medicine, 33 (1), 35-45

LEPR Gln223Arg exists in African population by Association with TB relapse not known.

Evidence: LEPRGln223Arg declines lung function and risk COPD

Nigeria : TB Epidemiological situation Parameter Value

Country Population 168,834,000 TB mortality, % (95%CI) 27 (1.6 -86) TB prevalence rate, freq (95%CI) per 100,000

persons (2012) 270 (43-710)

TB incidence rate, freq (95%CI) per 100,000 persons (2012)

180 (85 -310)

HIV/TB co infection rate, freq (95%CI) per 100,000 persons 2012 /2009

46 (21-80) /27

DOTS Population coverage (%) at at 2010 65 TB notification rate in 2010 (% new) 90,447 (58%) multidrug resistant Tb cases (New and Old (%) 2.2 and 9.4

TB Control : National TB and Leprosy Control Programme (Created, 1991).

STOP TB Partnership:

DOT plus + : sputum microscopy + HIV screening + Cat 1 Treatment at Designated DOT centers (>3,000) (Public + NTBLP affiliated private health facilities (PPP)

Treatment also provided by non-affiliated private health facilities

But re-treatment cases are presented for DR testing at reference centres such as NIMR (Few in the country).

TB Relapse: Nigerian context

•  Poorly documented like in many countries –  Limitations from contact tracing deficiency –  Lost to follow up –  Post-treatment monitoring (M & E gap) –  Patient’s relocation etc

•  Risk factors not well valuated among the few reported cases but treatments provided by non-affiliated NTBLCP health facilities implicated.

Specific Objectives

•  To establish the risk factors associated with TB relapse among affected TB patients in Lagos, Nigeria

•  To determine the allele and genotype distribution of LERP Gln223Arg among the study participants

•  To evaluate association between leptin receptor Gln233Arg Polymorphism and TB relapse in the patients.

METHODS

•  Study design : Case – control •  Setting: Lagos: one of the top 3 highest TB burden

states in Nigeria.

Nigeria Tuberculosis factsheet: http://nigeria.usembassy.gov

Methods Contd. •  Study Population:

Case: 25 TB relapsed patients (mean age + SEM, 34.2 + 1.4y) confirmed by sputum smear microscopy or X-ray or culture from 3 private health facilities, presenting for drug resistance screening at NIMR between Nov 2011 and March 2012

Control : 44 age-matched (mean, 32.9 + 1.6y) successfully treated TB patients@ 6 – 12 mo post treatment at DOT centres in Lagos

-  Inclusion criteria: HIV seronegativity (after 2 HIV tests)

-  absence or no history of diabetes (FBS < 105 mg/dL) -  no history of obesity, hypertension, surgery, pregnancy in the -  previous 3 mo -  willingness to participate/respond to questionnai

-  Ethical Approval: Lagos State Hospital Management Board (Ethics Committee)

Methods Contd. •  Risk factor measures:

–  Epidemiology: History of TB contacts –  Socio-Economic: Residence, Education, Occupation –  Biological : Age and Gender –  Lifestyle: Smoking –  Malnutrition: BMI < 18.5 kg/m2 –  Clinical : Fever (Ax > 37.50C); Anaemia (Hb < 11 g/dL)

Laboratory assays Blood collection: aliquots in plain tubes (Immunochemistry) EDTA tubes (DNA extraction + LEPR genotyping).

Immunochemistry : serum TNF-a and Leptin determination by Sandwish ELISA (Inter Assay CV 4.5 – 72%); Sensitivity Leptin > 0.5 ng/mL; TNF-α = > 5pg/mL

Methods Contd.

•  LEPR Gln 233Arg Genotyping (Ragin et al, 1999)

Primer Amplification Condition

Size Msp1 RFLP products, bp

F: 5′ACCCTTTAAGCTGGGTGTCCCAAATAG-3′ R: 5′-CAATATTTATGGGCTGAACTGACATT-3

95 deg, 5 min 95deg, 1 min 57.4deg, 1 min 72deg, 1 min x 35 72 deg, 10 min

330 bp W = 330 (AA) H = 330 + 293 + 37 M = 293 + 37 (GG)

•  Electrophoresis 0n 2% agarose gel

•  Statistical analysis : Descriptive statistics : for categorical and continuous variables •  Adjusted Odd ratio used to measure association of risk factors and LEPR Gln223Arg

polymorphism with TB relapse •  P < 0.05 was significant

RESULTS

Characteristics TB relapsed N = 25

TB recovered N = 44

Adjusted OR (95% CI)

Gender, n(%) Male Female

18 (72) 7 (28)

28 (63.6) 16 (36.4)

1.32 (0.31-4.2)a 1

Age, years Mean + SEM Median (Range)

34.3 + 1.4 34 (23 – 46)

32.9 + 1.6 30 (21 – 45)

> 0.05 > 0.05

Age group, n(%) < 40 years > 40 years

9 (36) 16 (64)

29 (65.9) 15 (34.1)

1 3.44 (1.1 – 11.0)*a

Educational status 1Primary Secondary Tertiary

4 (16) 16 (64) 5 (20)

7 (15.9) 29 (65.9) 8 (18.2)

0.88 (0.11 – 6.27)a 0.88 (0.21 – 3.78)a 1

Table 1. Sociodemographic and clinical characteristics of the TB patients and control

*P<0.05 (TB relapsed vs. TB recovered). a = adjusted by gender; b = adjusted by age; c = adjusted by residence

Results.. Characteristics TB relapsed

N = 25 TB recovered N = 44

Adjusted OR (95% CI)

Occupation Working Not working

17 (68) 8 (32)

27 (61.4) 17 (38.2)

1 0.71 (0.22 – 2.18)a

Smoking in the last 6 months Yes No

1 (4) 24 (96)

4 (9.1) 40 (90.9)

1 2.4 (0.23 – 58.8)

BMI, kg/m2, n(%) < 18.5 18.5 – 24.9 25 – 29.9

16 (64) 9 (36) 0 (0)

6 (13.6) 38 (86.4) 0 (0)

10.9 (2.8 – 40.3)*a,b 1

Axilliary Temperature, 0C Mean Median (Range)

37.4 + 0.06 37.2 (36.8 – 38.4)

37.1 + 0.03 37.1 (36.8 – 37.6)

*P<0.05 (TB relapsed vs. TB recovered). a = adjusted by gender; b = adjusted by age; c = adjusted by residence

Table 1. Contd

Results.. Characteristics TB relapsed

N = 25 TB recovered N = 44

Adjusted OR (95% CI)

Fever Yes No

9 (36) 16(64)

2 (18.2) 42 (81.8)

11.8 (2.1 – 89.5)*a 1

Smoking in the last 6 months Yes No

1 (4) 24 (96)

4 (9.1) 40 (90.9)

1 2.4 (0.23 – 58.8)

Hb, g/dL Mean + SEM Median (Range)

12.4 + 0.1 12.7 (10.3 – 13.2)

12.8 + 0.3 12.6 (11.7 – 13.5)

Anaemia Yes No

7 (28) 18 (72)

2 (18.2) 42 (81.8)

7.9 (1.1 – 63.4)*a 1

*P<0.05 (TB relapsed vs. TB recovered). a = adjusted by gender; b = adjusted by age; c = adjusted by residence

Table 1. Contd

Results..

Characteristics TB relapsed N = 25

TB recovered N = 44

Adjusted OR (95% CI)

Residence Urban slum Urban

14 (56) 11 (44)

27 (61.4) 17 (38.6)

0.74 (0.23 – 2.24) 1

History of TB contact Yes No

3 (12) 22 (88)

5 (11.4) 39 (88.6)

1.05 (0.14 – 5.37)c 1

Having TB was also not associated with history of TB contact. However, TB relapsed was found to be associated with age > 40 years, persistent anaemia, incidence of fever and low body weight after adjustment for gender and residence [OR, 3.4 – 11.8 (1.1 – 89.5), P< 0.05]. Smoking habit was reported by 4% and 9.1% of the studied groups

Table 1. Contd..

Results.. Table 2. Serum leptin and leptin receptor polymorphism among the TB patients. Parameter TB relapsed TB recovered P, OR (95% CI)

Leptin, ng/mL Mean + SEm Median (Range) TNF-a, pg/mL Mean LEPR Q223R polymorphism Genotype GG GA AA Allele G A Calculated χ2 Hardy-Weinberg Equilibrium

2.21 + 0.3 1.7 (0.6 – 6.2) 27.2 + 2.0 3 15 7 21 29 1.32 Yes

4.05 + 0.4 3.3 (2.1 – 8.6) 43.9 + 2.8 1 22 21 24 66 2.89 Yes

< 0.01 <0.01 < 0.01 0.04, 9 (0.63 – 268.68) 0.19, 2.1 (0.62 – 6.93) -, 1.0 0.06, 1.99 (0.90 – 4.41) -, 1.0

Although the distribution of the minor (G) and major (A) alleles of LEPR was in Hardy –Weinberg equilibrium in both study populations, homozygous inheritance of the minor allele in LEPR was associated with TB relapse

Parameter TB Relapsed TB recovered TB relapsed vs. TB recovered Carrier Non-carrier Carrier Non-carrier C vs. C NC vs. NC N = 18 N = 7 P N = 23 N = 21 p P-value P-

Age > 40 years BMI < 18.5 kg/m2, n (%) Fever, n (%) Hb, g/dL, mean + SEM Anaemia, n (%) @Leptin, ng/mL, Mean + SEM TNF-a, pg/mL, mean _ SEM

11 5 0.62 7 8 0.59 0.049 0.13 14 2 0.02 4 2 0.67 <0.0001 0.87 6 3 0.66 1 1 1.0 0.01 0.02 11.1 + 0.3 10.6 + 0.5 0.43 12.3 12.6 0.1 0.009 0.005 4 3 0.36 2 0 0.48 0.2 0.001 1.85 2.85 0.10 3.4 3.9 0.21 0.03 0.04 24.8 35.6 0.10 44.6 43. 5 0.8 0.01 0.04

Table 3. Effect of leptin receptor Q223R polymorphism on nutritional,

clinical and leptin level among the TB exposed patients.

Further analyses revealed significant association of low body weight but not serum leptin among TB relapsed but not TB recovered patients carrying the mutant allele of LEPRGln223Arg polymorphism.

Relationship Patients

TB relapsed (N = 25) r, P

TB recovered (N = 44) r, P

Total (N = 69) r, P

Leptin vs. BMI Leptin vs. Hb Leptin vs. TNF-α

0.61, < 0.05 0.22, > 0.05 0.18, > 0.05

0.58, < 0.05 0.63, < 0.05 0.54, < 0.05

0.57, < 0.05 0.36, > 0.05 0.31, > 0.05

Table 4. Correlation of serum leptin level with those of tumor necrosis factor-a, haemoblobin and body mass index between TB relapsed and TB recovered patients..

With the exception of BMI, the significant positive correlations between serum leptin and BMI, haemoglobin level and TNBF-α found in TB recovered patients were lost in TB relapsed group.

Discussion •  In this study Age, gender, rural residence were not found to be risk factors

for TB among the TB relapse.

•  This is contrary to studies in Asia where age > 40 y was a factor for TB relapse (Santha et al, 2002) or a study in Brazil where rural residence and lack of access to family health physician determined TB relapse (Batista et al, 2008; Oliveira et al, 2000)

•  In this study, persistent anaemia, decreased leptin and TNF-α level and homozygous mutant genotypes of LEPR Gln223Arg polyphorphism resulted in TB relapse

•  This study has provided an insight into the possible role of LEPR Gln223Arg polymorphism in treated TB patients’ susceptibility to TB relapse with a mechanism that is based on reduced weight gain, decreased systemic leptin and TNF-α

Discussion •  Our findings regarding reduced leptin and BMI in our TB relapsed patients

compared witrh TB recovered agree with the mechanism proposed by Cravel et al (2002) and Miller et al, (2000) but opposed that of Cakir et al, 1999.

•  Serum leptin relationship with poor weight gain after crisis in Nigerian sickle cell patients has previously been documented (Iwalokun et al, 2012).

•  However, that LEPR mutant genotypes is associated in our TB relapse patients with reduced serum leptin level needs for investigation since this polymorphism has been found to increase serum leptin level in obse and hypertension patients (Fan and Say, 2014; Itoh et al, 2002)

•  Our finding further supported previous works in which persistent anaemia and inadequate body weight were found to predict TB relapse strongly (Dooley et al, 2011)

Discussion •  The genotypes of LEPR Gln223Arg polymorphism in this study are in HWE

•  This is similar to previous studies among populations with African ancestry (Pena et al, 2014)

•  Our findings regarding reduced TNF-α as a predictor of TB relapse is not unexpected since protective mechanism against active TB in the lungs through alveoli macrophages and mobilized T- lymphocytes are TH1 dependent (TNF-α, IFN-γ, IKL-12, C3) to stable granuloma formation and microbicidal killing of the phagocytosed MTB in the lungs, maintaining a latent infectious state (Chan et al, 1992; Flynnet et al, 1995).

•  Therefore, based on the above, TB relapse may be pathologicala consequence of reduced TNF-α

•  Nevertheless, this study is limited by small sample size, inability to detect other polymorphisms of LEPR circulating in blacks and lack of data on other MTB protective TH1 cytokines.

Conclusion •  LEPR Gln223Arg polymorphism seems to modulate susceptibility to TB

relapse through impaired body weight and persistent anaemia among Nigerian patients

Future work. •  Larger study involving other ethnic groups •  Genome wide sequence approach •  Cellular immunology •  Inclusion of health system factors.

Co-Investigators and Affiliations

Names Affiliation Hodonu, S Mercy Hospital, Aina, AO Mayo Clinic Nwokoye, N NIMR Raheem, TY NIMR Banjoko, E Jaiyeola Hospital Nwaokorie, F NIMR Onibogu, C NIMR Idigbe, EO NIMR

ACKNOWLEDGMENT

Professor Oyedeji Kolawole: University of Lagos, Nigeria

Professor Oluwadun, Afolabi: Olabisi Onabanjo University, Nigeria

Professor Idigbe Oni: NIMR

Professor Ujah Innocent, mni NIMR

Dr. Sunday Alabi: Gabon

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References

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