IL1B gene variants, but not TNF, CXCL8, IL6 and IL10, modify the course of cystic fibrosis in Polish patients.

Background: The main aim of this study was to evaluate whether selected polymorphic variants in genes from the inflammatory pathway can be predictors of pulmonary or digestive manifestation of cystic fibrosis, as well as of severity of lung disease. Materials and methods: Using pyrosequencing and sequencing we have genotyped 12 variants in TNF (rs361525, rs1800629), CXCL8 (rs4073, rs2227306, rs2227307, rs188378669), IL1B (rs16944, rs1143634, rs1142639, rs1143627), IL6 (rs1800795) and IL10 (rs1800896) genes in a cohort of 55 Polish patients with diagnosed cystic fibrosis and controls. In our study group, a pulmonary manifestation of disease revealed 44 of subjects (80%), and digestive symptoms dominated in 11 (20%) of analyzed individuals. Severe lung dysfunction has occurred in 20 (36.4%) of patients. Results: We proved, that two promoter variants of IL1B, rs1143627 (c.-118G > A) and rs16944 (c.-598T > C) are presented significantly more often in patients with severe character of lung disease compared to mild (82.5% vs. 62.8%, p-value 0.030, and 87.5% vs. 64.3%, p-value 0.008, respectively) in cystic fibrosis course. Haplotype AC formed by both changes had also a higher frequency (80%) in patients with severe course compared to the mild character (61.4%) of disease. However, the frequency of promoter variant TNF c.-308C > T (rs1800629) was presented at a significantly lower level in the patient’s group compared to healthy controls (2.7% vs. 15%, p-value 0.001). Furthermore, the presence of methicillin-resistant Staphylococcus aureus significantly correlated with the lower FEV1% in patients (p-value 0.01). Conclusions: Genetic variants, rs1143627 and rs16944, of IL1B are promising candidates as predictors of the severe character of lung disease in Polish patients with cystic fibrosis.


Introduction
2][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. 4Today, over 2000 different CFTR mutations have been reported and F508del is by far the most common. 5Although 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. 6However, 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. 710] 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. 11Thus, 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. 8,12The 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. 14,17Furthermore, 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. 18,19Whereas, in French and German pediatric CF patients those results were not shared. 8sults 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.

Patients and clinical data
The study was approved by the local Ethics Committee of the University of Medical Sciences in Poznan, Poland (resolution no.675/15), and all experiments were performed following the relevant guidelines and regulations of this Committee.Written informed consent was obtained from each patient.55 Polish patients (20 males and 35 females) between the ages of 20-52 with diagnosed CF were enrolled for this study.The patient group was collected in 12 months (from January to December 2016) in the Department of Pulmonology, Allergology and Lung Oncology of the Clinical Hospital of Poznan University of Medical Sciences in Poland.Diagnosis of CF in all patients was REVISED Amendments from Version 2 Version 3 of this manuscript additionally include information about age of patients classified to severe and mild phenotype of the disease.Furthermore, the reference numbered 21 and the text was added to clarify the disease severity classification of patients.We also provided information about CF genotypes among patients group in the Table 4 containing analysed polymorphisms.Additionally, the information that patients were clinically stable at the time of study recruitment was added.
Any further responses from the reviewers can be found at the end of the article performed by sweat chloride test results (> 60 mmol/L) or/and identification of CFTR gene mutations.Detailed information about each patient including sex, age, BMI, age of diagnosis, presence of F508del mutation, pulmonary function parameters, function of internal organs, complications, and hospitalizations were recorded.Additionally, a control group of 50 healthy individuals was collected.A detailed characteristics of the study cohort with clinical and demographic data are presented in Table 1.
Pulmonary function tests, using Jaeger MasterScreen system (Erich Jaeger GmbH; Würzburg, Germany) were performed to assess lung function.All spirometric examinations were carried out with the subject seated, using a nose clip and a  20 At the same time the body plethysmography for assessing residual volume (RV), total lung capacity (TLC), and diffusing capacity of the lungs for carbon monoxide (DLCO) were performed.
Patients were divided in the context of lung function impairment, based on the FEV1% values, while they were clinically stable, 1 -within the norm (FEV1% ≥ 70) and mild pulmonary obstruction (FEV1% 40-70) (35 subjects in total), 2severe pulmonary obstruction (FEV1% ≤ 40) (20 subjects in total). 21The "severe" group of patients did not differ significantly in age from patients in the "mild" group (mean age was 27.11 and 30.75, respectively; p-value was 0.055).
In an attempt to analyze the correlation between the genotype and manifestation of CF, patients were divided into two subgroups depending on the dominant symptoms -the group with the manifestation primarily from the respiratory system (44 individuals) and the group of patients with prevalent gastrointestinal symptoms (11 individuals).The division was made by the specialists from the pulmonology field conducting the patients, based on the clinical data and interview.To the digestive predominant phenotype were enrolled patients with the coexistence of at least two of listed conditions: 1) diabetes or glucose metabolism impairment, 2) pancreatic insufficiency, 3) liver disease or cirrhosis, 4) nagging pain or dysfunction of the digestive system.All patients with GI predominant phenotype represented "mild" lung impairment.Specialists determining the patients phenotype were not involved in the analysis of genotype assessment.Results of genotyping did not influence the assessment of phenotypic description.
Pyrosequencing was performed by the PSQ™ 96MA system (Qiagen) using PyroMark™ Gold Q96 Reagents (Qiagen GmbH, Hilden, Germany), according to the manufacturer instructions.Direct sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) on the Applied Biosystems 3500 and Series Genetic Analyzers.

Statistical analysis
Conformance of genotypes distribution of all analyzed polymorphisms with the Hardy-Weinberg equilibrium (HWE) was assessed using Fisher's exact test.The pair-wise linkage disequilibrium (LD) of variants located in genes TNF, CXCL8, and IL1B was evaluated by Lewontin's D 0 using Haploview software version 4.2.The correlation analyses between genotypes and clinical data were performed using the chi-square test and Fisher's exact test.
For all calculations, STATISTICA 12.0 software (Stat Soft, 2014) was used.The level of significance was set at p < 0.05.

Allele frequencies and linkage disequilibrium analysis
A total of 55 CF Polish patients and 50 healthy controls were successfully genotyped for selected 12 polymorphisms located in genes CXCL8, TNF, IL1B, IL6, and IL10.Genotypes distribution for all SNPs met the requirements of HWE.No relevant differences in variant allele frequency between both groups were demonstrated.Only TNF c.-308C > T variant was observed significantly less often in the patient group (2.7%) compared to controls (15%), where the p-value was 0.001.All obtained frequencies of each genotype and allele are presented in Table 3.
Considering the fact, that analyzed changes formed in our study two haploblocks (in CXCL8 and IL1B genes), an analysis of the haplotypes in the context of the course and manifestation of disease was performed.We proved, that only haplotype AC created by changes c.-118G > A and c.-598T > C in IL1B gene is significantly more often observed in group with severe course of CF in comparison with mild course (80% and 61.4%, respectively; χ 2 = 4.055; p = 0.03).

Discussion
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.3][24] 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. 25Our 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. 26

Conclusions
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.

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

Is the study design appropriate and is the work technically sound? Partly
Are sufficient details of methods and analysis provided to allow replication by others?Partly If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Major comments:
The population size is relatively small for a study of this type and runs the risk of type 2 error (falsely exclude the null hypothesis).

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The definition of severe lung disease is based purely on lung function.However, this does not take into account age, i.e. a patient who has an FEV1 of 50% at the age of 20 years of age has a more severe phenotype compared to a patient who has an FEV1 of 50% of predicted at the age of 50 years (it is likely the 50 year old had a substantially higher FEV1 %predicted when they were 20 years old) 1 .There are other tools available in the literature for classifying the severity of lung disease taking age into consideration.

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Lung function and nutrition in people with CF are not independent.Often patients with the most severe lung disease, also have the worse nutritional status.Greater detail needs to be provided on how patients were classified to a pulmonary or GI predominant phenotype.This classification is subject to bias.It should be stated whether the specialists determining the patients phenotype were also involved in analysing the results of genotype assessment and whether all phenotyping was completed before the genotype results were available.In the future study, we will be careful and meticulous in classifying patients with CF to take age into account.
Lung function and nutrition in people with CF are not independent.Often patients with the most severe lung disease, also have the worse nutritional status.Greater detail needs to be provided on how patients were classified to a pulmonary or GI predominant phenotype.This classification is subject to bias.It should be stated whether the specialists determining the patients phenotype were also involved in analysing the results of genotype assessment and whether all phenotyping was completed before the genotype results were available.

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Thank You for this important remark.Of course, nutritional status is correlated with the severity of the disease.We can observe (based on BMI) this association in our study -in the "mild" group 26% of patients were malnourished, while in the "severe" group, 60% of patients.
Patients were classified by the specialists from the pulmonology field conducting the patients, based on the long-term observation of the patient and personal clinical interview with patient, to a pulmonary or GI predominant phenotype (we did not designate groups of pulmonary or digestive type of the disease, we only assessed that the dominant symptoms in a given patient were from the respiratory or digestive system, which does not mean that it was not from the other system).To the GI predominant phenotype were enrolled patients with the coexistence of at least two of listed conditions: 1) diabetes or glucose metabolism impairment, 2) pancreatic insufficiency, 3) liver disease or cirrhosis, 4) nagging pain or dysfunction of the digestive system.All patients with GI predominant phenotype represented "mild" lung impairment.Specialists determining the patients phenotype were not involved in the analysis of genotype assessment, of course.Results of genotyping did not influence the assessment of phenotypic description.
Therefore, in the text on page 6, the additional information was added to clarify: "To the digestive predominant phenotype were enrolled patients with the coexistence of at least two of listed conditions: 1) diabetes or glucose metabolism impairment, 2) pancreatic insufficiency, 3) liver disease or cirrhosis, 4) nagging pain or dysfunction of the digestive system.All patients with GI predominant phenotype represented "mild" lung impairment.Specialists determining the patients phenotype were not involved in the analysis of genotype assessment.

Results of genotyping did not influence the assessment of phenotypic description".
Information should be provided on the rates of the different polymorphism among people with different CF genotypes.If certain polymorphisms are over represented in people homozygous for the F508 mutation.It may be the CF genotype and not the polymorphism that explains the difference in phenotype.

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Thank You for this valuable comment.Accordingly to Your suggestion, we provide information about CF genotypes among patients group in the

Tadeusz Przybyłowski
Department of Internal Medicine, Pulmonary Diseases and Allergy, Medical University of Warsaw, Warsaw, Poland This is a very interesting paper in which the authors decided to evaluate whether selected polymorphic variants in genes encoding selected inflammatory mediators can predict pulmonary or digestive manifestation of cystic fibrosis and severity of lung involvement.Using advanced genetic engineering techniques they have genotyped 12 variants in TNF, CXCL8, IL1B, IL6 and IL10 genes in a group of 55 Polish patients with cystic fibrosis and 50 healthy controls.The most important outcome was the finding of significant differences in the frequency of two promoter variants of IL1B, rs1143627 (c.-118G > A) and rs16944 (c.-598T > C) in relation to the severity of lung involvement.It was also observed that AC haplotype was significantly more frequent in the group of patients with more advanced lung damage than in the group with milder disease progression.These results allowed the authors to conclude that genetic variants, rs1143627 and rs16944, of IL1B may be used as predictors of more severe respiratory involvement in the course of cystic fibrosis.Additionally, it should be noted that this is the first study of its type performed in a group of Polish patients.
The work was prepared very carefully, but in my opinion, 2 issues need re-evaluation.
The way of classifying patients into different stages of disease severity on the basis of FEV 1 is not very clear to me.Why the cut-off points of 70% and 40% of predicted were used?.In this part of method description there should be information if it was arbitrary classification or if it is consistent with the generally accepted system of grading, and in this case reference to an appropriate publication should be given.

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The paragraph concerning the occurrence of PA and MRSA in the studied group does not fit into the subject of the paper, does not add any new information, and may be omitted in the final version.Reviewer Expertise: Lung diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
were used?.In this part of the method description there should be information if it was arbitrary classification or if it is consistent with the generally accepted system of grading, and in this case reference to an appropriate publication should be given.
The paragraph concerning the occurrence of PA and MRSA in the studied group does not fit into the subject of the paper, does not add any new information, and may be omitted in the final version.

1.
The paragraph concerning the occurrence of PA and MRSA was omitted.

2.
Thank You very much.We hope our revised version of the manuscript will be correct now.
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*
Primers labelled with biotin for pyrosequencing.

(
EU population) IL1B c.-598T > C (rs16944Weinberg equilibrium (occurs when p > 0.05).Association analysis of clinical data and genetic diversity First, we have analyzed all 12 variants in designated, based on clinical data, groups of patients -with different manifestations of CF (with pulmonary or digestive dominant symptoms) and with variable courses of disease (mild or severe) to examine possible association.Our study demonstrated that the presence of two polymorphisms, c.-598T > C (rs16944) and c.-118G > A (rs1143627), in IL1B gene significantly correlate with character of disease (Table

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Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.

Table 1 .
Baseline characteristics of study participants.

Table 3 .
Genotypes and alleles distribution of analyzed polymorphisms among group of Polish CF patients and controls.

Table 3 .
Continued 1908C > 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.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]),whilealleleA 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.18They 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.19have 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.
8n contrast to those results, Corvol et al.8did 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.

Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results?
Information should be provided on the rates of the different polymorphism among people with different CF genotypes.If certain polymorphisms are over represented in people homozygous for the F508 mutation.It may be the CF genotype and not the polymorphism that explains the difference in phenotype.Lung function will vary with clinical status.Were all patients clinically stable at time of recruitment?If not, best lung function in the last 12 months may be a better marker of severity of lung function impairment.Yes "The "severe" group of patients did not differ significantly in age from patients in the "mild" group (mean age was 27.11 and 30.75, respectively; p-value = 0.055)".

Table 4
○ Thank you very much for this valuable remark.Of course, all the patients were clinically stable at the time of study recruitment.Reviewer Report 16 May 2022 https://doi.org/10.5256/f1000research.122087.r135127© 2022 Przybyłowski T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.