Cystic fibrosis (CF) is an autosomal recessive genetic disorder. The basic defect in the cystic fibrosis transmembrane regulator (CFTR) gene disrupts the function of the cystic fibrosis transmembrane regulator protein. CFTR encodes a chloride-conducting transmembrane channel, which regulates anion transport and airway mucociliary clearance [1]. Several bodily systems are thus affected. Comorbidities caused by the functional failure of CFTR occur in the pancreas (malabsorption and CF-related diabetes), liver (biliary cirrhosis), and sweat glands (heat shock), and can also result in infertility.

The major cause of morbidity and mortality in CF is the progressive pulmonary destruction, mostly due to a leukocyte-driven inflammation in CF lung disease [2]. Previously, we were able to show in F508del homozygous patients that there was a negative correlation between blood cytokines, such as transforming growth factor β1 (TGBβ1), and pulmonary function [3, 4, 5].

In CF patients with the same CFTR genotype (e.g., delta F508 (homozygous) [6, 7]), there are wide variations in the lung disease phenotypes [8, 9]. Large studies analyzing modifier genes provided evidence indicating that these modifier genotypes were more important for the pulmonary phenotype than the CFTR genotype. Severe pulmonary disease in F508del homozygous patients was associated with high TGBβ1-producing alleles [10]. These patients have increased levels of TGBβ1 in biomaterials, such as bronchoalveolar lavage (BAL) fluid (F) [11] and blood [12, 13], which is linked to fibrosis, goblet cell hyperplasia, abnormal inflammatory responses, and dysregulated ion transport.

The molecular mechanism of how TGBβ1 modifies CF lung disease remains unclear [11]. It has been shown by studies in vitro using human airway cells that TGBβ1 downregulates CFTR [14, 15, 16]. However, the relevance of these findings to CF, where CFTR activity might be minimal or completely absent is interestingly unclear. The relative impact of TGBβ1 expression on critical aspects of lung pathology in the presence and absence of CFTR expression remains unknown [11].

Recently, we demonstrated that carriers of the A allele with a single nucleotide polymorphism (SNP), rs41266431, in the gap junction protein alpha 4 (GJA4) gene are better protected against end-stage lung disease (ESLD) and produce better lung function tests. The explanation is that this SNP causes an amino acid substitution [17]. Moreover, this variant has no effect on the frequency of acquiring Pseudomonas aeruginosa (P. aeruginosa). However, CF patients chronically colonized with P. aeruginosa, and carriers of the A allele have significantly better pulmonary function than those with the G/G genotype.

CFTR modulator therapies targeting the basic defect in CF and restore CFTR function are currently available. Moreover, they can effectively improve nutritional status, as well as the quality of life, via lung function and a reduction in pulmonary exacerbations. Therefore, the question arises, as to whether there remains a need for further genotyping of disease-modifying variants. The most successful CFTR modulator Trikafta (elexacaftor, ivacaftor, and tezacaftor) was firstly licensed in 2019; however, data are lacking as to the evidence of a long-lasting effect ($>$10 years). Similarly, for Ivacaftor, e.g., the effect on the body mass index (BMI) was no longer statistically significant after 5 years [18]. Moreover, we cannot evaluate the long-term side effects (e.g., to the liver) of using a triple modulator therapy. Thus, precise genotyping might be needed to prevent any unnecessary use in patients, e.g., those exhibiting a mild clinical phenotype but at high risk of liver disease. In addition, there are also countries with a moderate healthcare plan/budget. Precise genotyping to predict the clinical phenotype could help to decide the patients that should be prioritized for treatment with an expensive CFTR modulator.

The aim of this paper was two-fold: Firstly, to examine the impact of a well-documented pulmonary CF modifier, TGBβ1 [10, 19], on clinical phenotype in F508del homozygous CF patients in a large German cohort and in the initial Dutch cohort. Secondly, to test for a potential co-modulation of the GJA4 and TGBβ1 genotypes on the clinical disease phenotypes.

We demonstrated that GJA4 variants are independent of TGBβ1 variants. Both variants had an impact on the lung function tests (LFTs), although only GJA4 variants were associated with a better outcome for death as well as for death and ESLD.

Concerning gene–gene interactions, the only haplotype that had a significant impact on survival was the haplotype TGBβ1 carriers of C + GJA4 G/G. However, in the Cox regression analysis, there was no significant augmentation of risk to death, similar to GJA4.

Analysis of the overall cohort revealed a significantly lower lung function was linked to the TGBβ1 (FEF75), as well as the GJA4 (FVC) risk variant. Though evaluating haplotypes and extreme risk haplotypes (see Supplementary Material) for gene–gene interactions did not show any augmentation of these effects. Observation of those chronically colonized with P. aeruginosa (Table 2) revealed a significantly lower lung function in the TGBβ1 carriers of C + GJA4 G/G compared to the TGBβ1 carriers of C + GJA4 A/G. In this regard, it appears that in terms of lung function, the GJA4 genotype was more important than the TGBβ1 for those chronically colonized with P. aeruginosa.

Moreover, in terms of lung function, the high-risk haplotype was not associated with more severe results compared to the other risk variants (Supplementary Table 1). In those chronically colonized with P. aeruginosa, we were able to observe significantly improved lung function values (FEV1 and FVC) in patients with the low-risk haplotype compared to those at intermediate risk. As there was no significant difference between a high- and a low-risk haplotype, it should be discussed whether haplotype risk has an impact on the clinical disease phenotype.

Several studies with large patient numbers have shown that variants of the TGBβ1 gene have an impact on clinical phenotype in F508del homozygous CF patients [10, 19]. Most of this data was generated from CF populations in northern America [10]. Currently, there are fewer studies involving German patients [4, 22, 23], which present smaller numbers of F508del homozygous patients (n 30) and to the best of our knowledge, Dutch patients. Trojan and coworkers [22] could not find a significant association between lung function and the polymorphisms in the TGBβ1 promoter, which corresponds to our initial study [4]. This might be attributed to the low number of F508del homozygous patients in those previous studies. Corvol et al. [23] could only find an association for the +869 T/C variant but not for the –509 T variant (rs1800469). In this regard, our cohort, which indicated a significant impact of the –509 T variant (rs1800469) on lung function, represents the first study in German CF patients to confirm the data by Drumm and coworkers [10]. They used several models to interprete the –509 variant in the TGBβ1 promoter: The odds ratio was the best for the recessive model (comparison of the T/T variant with the combined C/T and C/C genotypes). In addition to the recessive model, we also evaluated the codominant (comparison of the T/T with the C/C genotype) and the dominant models (comparison between the combined C/T and T/T genotypes and the C/C genotype). Here, we were only able to find significant differences in lung function parameters in the recessive model.

The disadvantage of this is the low incidence (15%) rates of these variants, which could explain why the expected difference only reached statistical significance for FEF75 and not FVC. Moreover, with absolute numbers of only three patients that were homozygous for the T/T genotype of the –509 variant in chronically P. aeruginosa colonized patients (Supplementary Table 2), we doubt the relevance of a significantly better BMI in those with the risk variant in our study.

Among the 115 patients, there were 3 sibling pairs. In terms of the GJA4 genotype, all 3 were carriers of the protective A allele. For the TGBβ1 genotypes, all were carriers of the protective C allele (C/C genotype n = 5, C/T genotype n = 1). Therefore, in summary, the 3 siblings pairs were not carriers of a risk genotype. Therefore, given that the phenotype was also attributed to environmental factors, the question remains as to whether there were any differences in these sibling pairs. In detail, two pairs of siblings were chronically colonized with P. aeruginosa (18/15 y; 50/47 y). Among those, one male patient received a lung transplant, while the younger sibling of 3 years had a FEV1 of 78% predicted. The extreme differences can be attributed to compliance. For the second pair of siblings with P. aeruginosa colonization, the brother, who was again 3 years younger, had an approximately similar FEV1 (65 vs. 63% predicted). The third pair (18/15 y) also showed some differences (FEV1 80/90% predicted). This can be attributed to the older sibling having intermittently P. aeruginosa, meaning the compliance was slightly better in the younger siblings. In this regard, our data are in keeping with a study on Italian CF sibling pairs [24], which reported on the heterogeneity in CF siblings with the same CFTR genotype. Moreover, our data also support these findings for two CF modifier genes (GJA4/TGBβ1).

In terms of serum TGBβ1, we could not confirm the negative correlation between serum TGBβ1 and lung function, which we found in our previous study [4] or, which was later presented in a German study [22]. Those studies with small numbers of F508del homozygous patients (n 30) were conducted with younger patients (median age: 20 y [4]/21 y [22]) and fewer patients. We assume that our cohort (mean age: 27 y) with a higher rate of chronic P. aeruginosa colonization (50%) compared to those studies (Trojan et al. [22]: 37.5%, Eickmeier et al. [4]: 33.9%) other proinflammatory mechanisms might be important: TGBβ1 does not affect the efficiency of phagocytosis [25, 26, 27, 28]. Moreover, TGBβ1 can be activated either by an $\alpha$$\nu$$\beta$6 or an $\alpha$$\nu$$\beta$8-mediated pathway. The $\alpha$$\nu$$\beta$6-mediated activation appears to be fundamentally dependent on direct cell–cell contact and does not release any diffusible free TGBβ1 [29]. Such a pathway is ideally suited to the alveolar space. Nevertheless, TGBβ1 activated by this pathway can have an impact on lung function and will be influenced by high-producer genotypes, although it will not be measurable in blood. The release of free TGBβ1 is provided by activation from the $\alpha$$\nu$$\beta$8 integrin in the conducting airways [30]. This mechanism does not depend on direct cell–cell contact and should provide TGBβ1 with the ability to diffuse and translocate, thereby affecting distant cells. Moreover, $\alpha$$\nu$$\beta$8 mRNA is expressed in a variety of leukocytes [31].

We could not provide evidence of a significant impact of the SNP-509 (rs1800469) on the TGBβ1 gene in end-stage lung disease (transplantation or death). This is in keeping with the data reported in previous studies; however, to the best of our knowledge, no study has previously reported on the impact of TGBβ1 variants on ESLD.

GJA4 gene variants are independent of TGBβ1 variants. Both variants have an influence on disease phenotypes relating to lung function, although only GJA4 variants are associated with better outcomes in ESLD.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

JPL: investigation, data curation, and writing–original draft and review. ML: conceptualization, methodology, investigation, resources, writing–original draft (lab part) and review, and supervision (genetic lab). TH: investigation (genotyping). OE: investigation (inflammatory markers) and writing, review, and editing. CS: investigation and writing, review, and editing. RS: investigation, methodology (inflammatory markers), supervision (lab), resources, and writing–review and editing. SZ: conceptualization (inflammatory markers), writing, reviewing, and editing. CM: investigation, writing, review, and editing. MA: writing, review, and editing. AS: analysis of the data, review. SS-G: conceptualization, methodology, data curation, formal analysis, funding acquisition, writing–original draft and review, supervision, and project administration. All authors have contributed to the manuscript and approved the submitted version. All authors have read and agreed to the published version of the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Studies involving human participants were conducted in accordance with the Declaration of Helsinki reviewed and approved by the University of Bonn (178/01 + 092/17), University of Frankfurt (07/02 + 206/16), and Amsterdam University Medical Center (NL60220.018.16). Informed consent was obtained from all subjects involved in the study.

Not applicable.

This research was funded by the heritage of Juliana Gerner.

Dr. Sabina Schmitt-Grohé is on the advisory committee of Vertex and has received a travel grant from Vertex (for ECFS 2019) and ERS (ERS Research Seminars). Moreover, she gave a talk to Vertex, participated in studies for the same, received a fee for a congress report (ECFS conference Liverpool 2019), and attended a vertex-sponsored conference (1.CF-Akademie, Schloss Hohenkammer). In addition, she got financial reimbursement for a talk for Deutsche CF Hilfe and received a personal fee for an expert opinion for the Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen. Dr. Stefan Zielen reports grants and personal fees from Bene-Arzneimittel GmbH, grants and personal fees from Biotest GmbH, grants from Vifor Pharma Deutschland GmbHÐ, grants from ALK Arzneimittel, personal fees from Novartis GmbH, personal fees from Böhringer Ingelheim, personal fees from Lofarma GmbH, personal fees from IMS HEALTH GmbH and Co., personal fees from GSK, Stallergen, Procter and Gamble, and Allergopharma GmbH, grants and Allergy Therapeutics, Engelhard Arzneimittel, Sanofi-Pasteur, AstraZeneca, Erydel, and Bionorica GmbH, outside the submitted work.