Recent scientific outcomes confirm that the clinical phenotype of cystic fibrosis (CF) (OMIM: 219700) is determined not only by classes of mutations in the CFTR gene (cystic fibrosis transmembrane regulator) but in association with environmental factors and genetic variations in modifier genes. ^{1 – 3} The hypothesis about the role of modifier genes in CF was born based on the observations, that patients with the same CFTR genotype presented diverse manifestations and course of the disease. ⁴ Today, over 2000 different CFTR mutations have been reported and F508del is by far the most common. ⁵ Although mutations in the CFTR gene are well known and classified, the contribution of modulatory genes in CF is currently still investigated. Among analyzed candidate genes are those involved in the inflammatory process, as well as in immunity and antioxidant molecules. ⁶ However, the results of the majority of global research on modifier genes’ role in CF are inconclusive.

CF is a multi-organ disease, whereas chronic pulmonary inflammation and respiratory failure consist of the main cause of death in those patients. There is evidence, that the inflammatory process in the lung is associated with an imbalance between pro- and anti-inflammatory mediators. ⁷ Among important pro-inflammatory cytokines produced during the response are tumor necrosis factor-alpha (TNF-α), interleukins (IL) 8, 6, 1, and 1B, while among cytokines inducing the opposite effect are transforming growth factor-beta 1 (TGFB1) and IL10. ^{8 – 10}

Proteins, IL8 and TNF-α, play a crucial role in the pathophysiology of CF lung disease due to their participation in the recruitment and activation of neutrophils on the respiratory epithelial surface, which is a primary component of the innate immune response. ¹¹ Thus, genes CXCL8 and TNF coding for those cytokines, which expression is regulated by sequence variants, are highlighted as potential modifier genes in the severity of lung disease in CF. Although numerous polymorphic variants have been described in the CXCL8 gene, the association only between polymorphisms rs4073 (c.-251T>A), rs2227306 (c.781C>T), rs2227307 (c.396T>G), pulmonary function, and clinical severity markers in CF patients was confirmed in several studies. ^{8 , 12} The most often analyzed changes in the TNF gene are located in the promoter region, such as c.-238G>A (rs361525) having a variable effect on gene expression and c.-308A>G (rs1800629) associated with increased gene transcription, worst pulmonary function, and early pulmonary symptoms in patients with CF. ^{13 , 14} in contrast to studies performed by Schmitt-Grohé et al. ¹⁵ and Khorrami et al. ¹⁶ There is also proven, that some of TNF and CXCL8 polymorphisms are associated with Pseudomonas aeruginosa ( PA) chronic colonization in CF patients. ^{14 , 17} Furthermore, modulating effects on the CF also have shown IL1B and IL10 genes, where the most common SNPs were associated with severe lung disease in pediatric American and Australian populations. ^{18 , 19} Whereas, in French and German pediatric CF patients those results were not shared. ⁸

Results of up to now performed studies, searching for genes that modify the course and phenotype of CF, mostly concern the association with pulmonary exacerbation. However, based on our long-term observations of CF patients we state, that around 20% of CF patients manifest a pronounced exacerbation of symptoms from the digestive system. We hypothesized, that this possibly may be predicted by polymorphic changes at immunologically relevant genes. Within this context, we have selected 12 polymorphisms located in five genes CXCL8 (rs4073, rs2227306, rs2227307, rs188378669), TNF (rs361525, rs1800629), IL1B (rs16944, rs1143634, rs1142639, rs1143627), IL6 (rs1800795), and IL10 (rs1800896) for correlation analysis, as candidate genetic modulators of the pulmonary or digestive manifestation and severity of the disease among Polish CF patients.

Because CF is a multifactorial, life-shortening disorder, the determination of SNPs that would affect the general phenotype or course of the disease is essential, but also challenging due to previous inconclusive results. So far, most of the CF studies were focused on searching modifier genes responsible for the severe pulmonary phenotype of the disease. In our investigation we have analyzed the impact of selected 12 potential candidates of modulator changes, rs4073, rs2227306, rs2227307 and rs188378669 in CXCL8 gene , rs361525 and rs1800629 in TNF gene, rs16944, rs1143634, rs1142639 and rs1143627 in IL1B gene, rs1800795 in IL6 gene and rs1800896 in IL10 gene on CF phenotype in Polish patients, taking into account the severity of symptoms on the side of the digestive, but also, respiratory system. Our hypothesis was that candidate modulator changes may predict digestive character of CF.

We observed, that in most of our group of patients (80%) the dominating symptoms occurred from the respiratory system and only in 20% of CF patients from the digestive system. Severe character of lung disease, diagnosed based on the FEV1% values, was noted in 20 patients (36%) and mild in 35 individuals (64%). In those subgroups of patients, we have performed a correlation analysis with DNA changes. Obtained variant allele frequencies of analyzed genetic variants, did not much differ from reference values for European population in 1000 Genomes database, except change TNF c.-308C > T (rs1800629) which occurred in our patients group less often (2,7%) than in the database (13%). Also interesting is, that variant CXCL8 c.91G > T, p.Glu31Ter (rs188378669) globally noted with variant allele frequency < 0.1%, was detected in our CF patients at level 0.9% (one heterozygote detected in a cohort of 55 individuals). In our previous study, we proved, that this variant is significantly more common in patients with inflammatory bowel disease (MAF = 2.12%, 15 heterozygotes detected in a cohort of 353 patients) compared to healthy Polish population (MAF = 0.25%, 1 heterozygote identified in a cohort of 200 individuals of Polish population), what may suggest its association with inflammatory diseases (unpublished data). Therefore, studies on a larger group of patients are undoubtedly necessary to verify the participation of this variant in CF, especially since there are no data on the relationship with this disease.

Our study revealed, that among all analyzed genetic changes two of them, c.-598T > C (rs16944) and c.-118G > A (rs1143627) located in the IL1B gene, are significantly associated with the severe character of lung disease in polish CF subjects. Allele C in locus -598 was observed with frequency of 87.5% in patients with severe lung disease compared to patients with mild lung dysfunction (64.3%, p = 0.008, OR = 3.88, C.I. = [1.351-11.190]), while allele A in locus -118 was observed with frequency 82.5% and 62.8% in both groups, respectively (p = 0.03, OR = 2.78, C.I. = [1.079-7.194]). We confirmed high LD between both changes (rs1143627 and rs16944) creating haplotypes AC, GT, GC and AT, where AC was significantly more often observed in subjects with severe course of CF in comparison to mild.

Our findings concerning the impact of polymorphism rs16944 on CF phenotype are consistent with those obtained by de Vries et al. ¹⁸ They also proved a significant correlation of the variant allele c.-598C of IL1B gene with severe pulmonary dysfunction in total of 152 Australian CF patients. Similarly, Levy et al. ¹⁹ have reported that IL1B constitutes a clinically relevant modulator of CF lung disease in the study conducted among American patients. However, in their research other SNPs, rs1143634 and rs1143639 demonstrated a consistent association with severe pulmonary phenotype.

In contrast to those results, Corvol et al. ⁸ did not find any correlation between variants c.-598T > C and c.-118G > A in IL1B gene and lung function assessed by spirometry in 329 Caucasian CF children from France and Germany. Additionally, they did not confirm any linkage disequilibrium between those polymorphisms.

Studies mainly highlight the relationship between lung disease in CF and CXCL8 gene polymorphism. ^{21 – 23} IL8 plays a crucial role in the pathophysiology of inflammation of the airways in CF patients caused by a deficiency or absence of the CFTR protein. ²⁴ Our study did not confirm this association among Polish patients.

We are aware of several limitations of our research. Our study cohort included only 55 patients and 50 controls. In the next step, verification of our results should be performed on a larger group of patients. This may be crucial in the case of rare variants, as c.91G > T, p.Glu31Ter (rs188378669) in CXCL8 gene, candidate as a modifier of CF. Furthermore, other factors such as BMI, gender, or age of diagnosis was not taken into account in our statistical analyzes.

We should also highlight the strengths of our study. First, the study cohort was represented by detailed characterized patients and homogenous controls group. What is important, the effect of CFTR mutation F508del on the manifestation and course of CF in the studied patients was excluded because the frequency of mutations in the subgroups was similar.

Although this study does not indicate any modulators of digestive manifestation of CF, it constitutes the first report of genes predicting the course of this disease in the Polish population.

Recent studies indicate the important role of the microbiome in the course and manifestation of cystic fibrosis. Scientists underline that both, genotype and microbiome profiles are crucial interconnected factors in disease progression. ²⁵

Our data have shown, that from all analyzed pro-inflammatory cytokine genes, only IL1B, but not TNF, CXCL8, IL6, or IL10 clearly play a crucial role in CF manifestation, determining the severe character of lung disease. This is a confirmation of major global results, as well as the first report concerning modulator genes of CF manifestation among Polish patients. Unfortunately, none of the analyzed genetic variants was found as predictors of digestive manifestation of CF disease, which may suggest the participation of also other modulator genes in the final phenotype of the disease.

All data underlying the results are available as part of the article and no additional source data are required.