In recent years, scientific interest has focused on the genetic study of predisposition as a prognostic factor for the course of tuberculosis. Numerous genetic polymorphisms have been examined in relation to their association with the infectious agent Mycobacterium tuberculosis.

Research has primarily concentrated on the following genes, which are involved in the immune response to Mycobacterium infection: CCL2 (chemokine ligand 2), NRAMP1 (natural resistance-associated macrophage protein 1), IRGM (immunity-related GTPase family), IL8 (interleukin 8), TLR (toll-like receptor), NOD2 (nucleotide-binding oligomerization domain-like receptor family), TYK2 (tyrosine kinase 2), and SP110 (nuclear body protein), among others.

Based on an extensive literature review [9] - [29] , we aimed to analyze the protective and predisposing potential of polymorphisms in the NRAMP1 gene, as described above, in patients from the Bulgarian population.

We collected 45 samples from 10 families. Twenty-seven of these were from children, and 16 were from parents. Among all participants, 13 individuals (both parents and children) were healthy, while 32 were either sick or had relapsed. Pediatric patients ranged in age from 1 to 16 years, with a mean age of 9 ± 5 years and a male-to-female gender distribution of 3:7.

Our sample size is 45 individuals. For the case-control study we chouse 10 children that are not relatives. For controls we chouse 50 healthy children with the same mean age and sex. Detection of significant associations is observed by determining odd ration and p values.

The disease was confirmed through clinical, radiological, and immunological tests conducted at the Clinic of Pediatric Pneumology and Phthisiology, University Hospital “St. Ivan Rilski,” Sofia.

Inclusion criteria

Families with children who were treated in our clinic in 2023, to whom the essence and benefits of the study were explained, and who gave their consent to be included. The families have different numbers of children. Some have an active form of tuberculosis, others with latent tuberculosis infection. Some of the parents are healthy, others have been ill or are currently being treated for tuberculosis.

All included with active tuberculosis, or treated for such, have had a clinical picture of tuberculosis: intoxication and consummative syndrome, auscultatory changes specific to the different forms of the disease, X-ray data, and positive immunological tests specific to the disease: Mantoux test, T-SPOT TB test, QuantiFERON-TBC Gold Plus.

The treatment is carried out according to the standards for the treatment of tuberculosis introduced by the World Health Organization. It is carried out in two phases: intensive and long-term. Those who have completed treatment are monitored for the next two years.

Exclusion criteria

Families with a child or parent with active tuberculosis or a history of tuberculosis refused to be included in the study.

The main forms of pulmonary involvement included tuberculosis of intrathoracic lymph nodes (TBITLV), primary tuberculosis complex (PTK), infiltrative-pneumonic form (IPF), one case of tuberculous meningitis (TM), and several cases of latent tuberculosis infection.

All participants provided informed consent for genetic testing.

A total of 10 ml of venous blood was collected into a 50 ml sterile conical plastic tube and mixed with 30 ml of cold lysis buffer, followed by incubation for 30 minutes on ice with occasional shaking. The mixture was then centrifuged at 3000 rpm for 15 minutes. The supernatant was carefully poured off and discarded, and the tubes were allowed to drain on filter paper.

The pellet was resuspended in 10 ml of cold lysis buffer and centrifuged again under the same conditions. After removing the supernatant, the pellet containing nucleated cells was resuspended in 5 ml of SE buffer. Next, 25 ml of proteinase K (10 mg/ml) and 250 ml of 10% sodium dodecyl sulfate (SDS) were added, followed by mixing and incubation at 37˚C overnight or at 55˚C for 5 hours.

After proteolysis, 1.4 ml of pre-saturated (6 M) sodium chloride (NaCl) was added, and the mixture was vortexed. Centrifugation was performed at 3000 rpm for 15 minutes at room temperature to prevent SDS precipitation. The DNA-containing supernatant was carefully decanted into a new 50 ml sterile conical plastic tube, and two volumes of ice-cold absolute ethanol were added. Under these conditions, the DNA precipitated visibly, floated to the surface, and was scraped off using a sterile (burnt) cloth. The DNA was washed in 70% ethanol, air-dried, and dissolved in sterile TE buffer.

The concentration and purity of the isolated genomic DNA were measured spectrophotometrically. This method relies on the specific absorption of light at a wavelength of λ = 260 nm by purine and pyrimidine bases in DNA. The ratio of absorbances measured at λ260 and λ280 indicates the purity of the DNA and should be between 1.8 and 2.0. Values below 1.8 indicate the presence of protein impurities, while values above 2.0 suggest RNA contamination.

The selected polymorphisms in the NRAMP1 gene for this study were INT4 (rs3731865), D543N (rs17235409), and 3’UTR (rs17235416).

The primers necessary for amplification of the indicated polymorphisms, were selected according to article [25] and are presented in Table 1 .

After a series of optimizations, a protocol was developed for the optimal components of the amplification reaction ( Table 2 ). The optimal conditions for amplification are presented in Table 3 .

The analysis is based on cutting DNA with specific restriction enzymes. Individual

fragments typically differ due to point mutations that either create or disrupt restriction sites or through deletions/insertions, which can alter the length of the restriction fragments. The restriction enzymes used and the RFLP reactions are presented in Table 4 .

Following incubation at 37˚C overnight (o/n) and visualization on a 3% agarose gel stained with ethidium bromide, the samples were analyzed using a molecular marker. The length of the amplified fragments was determined relative to the Quick-Load^® Purple 1 kb Plus DNA Ladder (NEB, USA).

Statistical analysis of the data was performed using SPSS-20 [30] , and case-control and family-based association analyzes were performed with Plink version 1.07 [31] . Plink is genome-wide association analysis software with focus on genotype/phenotype data analysis.

Of the NRAMP1 polymorphisms studied—INT4, D543N, and 3’UTR—both 3’UTR (1729 + 55del4) (rs17235416) and D543N (codon 543, Asp to Asn) (1627G/A) (rs17235409) exhibited normal genotypes.

For INT4 (469 +14G/C) (rs3731865), a total of 43 samples were analyzed. Among them:

In percentage terms, the distribution of the INT4 polymorphism was as follows:

The results of the family-based association analysis for INT4 indicated that the increased frequency of heterozygotes and homozygotes for the mutation was not statistically significant ( Table 5 , Figure 3 , and Figure 4 ).

VGLV—1 homozygote and 8 heterozygotes; PTK—1 heterozygote; IPF. 1 homozygote and 4 heterozygotes; ME—1 heterozygote; FCF—one homo- and heterozygote each, LTBI—one homo or heterozygote each. Of note is the fact that

2 homo- and 4 heterozygotes are also observed in the healthy.

In the most common childhood form, VGLV tuberculosis, 53% are heterozygotes, 6.6% homozygotes.

In infiltrative-pneumonic, one of the severe forms of tuberculosis, 80% of the included patients are hetero- and 20% are homozygous for the mutation.

Isolation of Mycobacterium tuberculosis by direct microscopy or culture is essential for determining the form and severity of tuberculosis. Of particular interest is the frequency of bacillus detection in relation to the genetic phenotype. Notably, in children, who represent a significant proportion of the studied group, bacillus separation was observed in only 20% - 25% of the examined cases.

Among all tested individuals with detected bacilli (n = 8), 50% were heterozygotes and 12.5% were homozygotes. The percentage of heterozygotes was notably high in samples from bacilli-secreting patients.

The results of the case-control association analysis further indicated that no statistical significance was found for the TB-predisposing effect of NRAMP1 INT4 ( Table 6 , Figures 5 - 7 ).