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

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.

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

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

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

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

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’

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

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

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.

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.

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

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.