1. Molecular study of two types of mutations in promoters of IL-2 and IL-10 genes in Iraqi patients with Tuberculosis
Mazin S.Salman Awatif H.Issa College of Sciences University of Basra / Iraq

2. Introduction
Tuberculosis (TB) is one of the infectious diseases, which is spread from one individual to another by air droplet.
Mycobacterium tuberculosis (MTB) is the main causative agent of TB.
Statistics indicate that one-third of the world's population is harboring a latent TB infection, approximately 1.5million deaths occurring annually from the active disease coming in second after human immunodeficiency virus(HIV) as a cause of death worldwide.

3. Introduction
Familial clustering data, animal models, twin studies and complex segregation analysis and many others studies clearly demonstrated that there is a genetic basis to accept the incidence of tuberculosis.
Cytokines broad and loose category of small proteins (~5–20 kDa) that are important in cell signaling.
Cytokines play a critical role in the defense against bacterial infections, both directly, by the inhibition of bacterial replication, and indirectly, through determination of the prevalent Th1/Th2 pattern of host immune response

4. Introduction
Interleukin-2 (IL-2) and Interleukin-10 (IL-10) among most important cytokines which playing great role as regulatory cytokines for host immunity against pathogens.
IL-2 gene located on chromosome 4 and it has a crucial role in generating an immune response by inducing lymphocytes specific for an antigen, so it called lymphokine.
IL-10 has critical role in bacterial infections , ). IL-10 can reduce the production on INF- γ so the increase in IL-10 levels appears to support the mycobacterial survival in the host this means the increase in IL-10 production can suppress the immune response

5. The main aim of present study is to find out the main types of mutations, which occurred in promoter region of two gene that coded for two of most important regulatory cytokines, which are IL-2 and IL-10 and possible correlation between these mutations and outcome of TB infections.
Aim of the study

6. Extraction of genomic DNA Genetic Analysis by BCR of
Material and Method
Patients
Blood
EDTA tube
Extraction of genomic DNA
IL-2 promoter
Genetic Analysis by BCR of
IL-10 promoter

7. Two sets of specific primers for promoter regions of IL-2 and IL-10 genes
Table (1): Primers using in PCR amplification of IL-2 and IL-10 gene.
Material and Method
Primers
Sequence
product size
Reference
IL-2
sense
5’-AAGAGTCATCAGAAGAGGAA-3’
150 bp
Sivangala et al.,2013
antsense
5’- AGCTGATCAGGTCCAAAGGA-3’
IL-10
5’-TTCCCCAGGTAGAGCAACAC-3’
565bp
Spinasse et al.,2012
atisense
5’-GGCACATGTTTCCACCTCTT-3’

8. Material and Method
Table (2): IL-10 promoter amplification conditions by PCR
Table (3): IL-2 promoter amplification conditions by PCR
Steps
Temperature
Time
Number of cycles
Initial denaturation
94°C
5 min 1
Denaturation
1 min 35
Annealing
63.3°C
Extension
72°C
Final extension
Soaking
4 °C
Indefinite
Steps
Temperature
Time
Number of cycles
Initial denaturation
95°C
5 min 1
Denaturation
30 Sec 30
Annealing
53°C
Extension
72°C
35 Sec
Final extension
10 min
Soaking
4 °C
Indefinite

9. Polymorphism in IL-2 promoter
Figure (1): PCR products of the DNA amplicons of IL-2
Table (4). The genotype and allele frequencies of the identified IL-2 SNPs
Locus IL-2
Genotype
Patients (n=74)
Healthy control (n=74)
Absolute Frequency
Allele frequency
p-value P.
-312 AA CA
f(C) 66 8 74
0.89
0.1
0.002
0.623
-330 TT GT
f(G) 58 16
0.79
0.19
0.01
0.2971

10. Polymorphism in IL-2 promoter
Figure (2) : (A) Alignment of sequences (A/C) allele appear with red letter at position -312.(B) alignment of chromatograms appear its peaks quality.

11. Polymorphism in IL-10 promoter
Figure (3): PCR products of the DNA amplicons of IL-10
Figure (4): The alignment of CA motifs associated with TB infection which appeared highly frequency in (CA) repeats.

12. Table (5): Frequency of (CA) n alleles in the I-10 gene in the TB patients and controls

13. Conclusion
There are more than one type of mutations pushing toward increasing susceptibility of some individuals to TB, mutation in promoter region of regulatory cytokines such as IL-2 and IL-10 plays critical role in altering immune response for the sake of pathogen.