The study was approved by the local ethics committee (Consent of Research Review Board at the Medical University of Lodz, Poland, No RNN/133/09/KE). At the commencement of the study, the participants were invited to get involved voluntarily. Before the enrolment, written informed consent was obtained from each patient.

Asthma diagnosis was established according to GINA (The Global Initiative For Asthma) recommendations, based on clinical asthma symptoms and a lung function test. The level of asthma severity and control was determined on the basis of the GINA Report Guidelines. All the participants underwent subjective examinations (structuralized anamnesis including, besides the element of subjective examination, also an analysis of factors such as: gender, obesity, tobacco smoking, duration of bronchial asthma, allergy to house dust mites, animal fur, mold spores, cockroaches allergens, hypersensitivity to non-steroid anti-inflammatory drugs (NSAIDs), etc. Results of pulmonary function tests and allergological tests were obtained from individual medical records of the patients. If results of spirometry or allergological tests were not available, such examinations were additionally performed during the recruitment visit. Subjects are suffering from clinically significant exacerbations and using drugs that might induce resistance to glucocorticoids (such as rifampicin, phenobarbital, phenytoin, ephedrine), subjects with signs of viral infections, either generalized or affecting the respiratory tract, as well as subjects failing to comply with the doctor’s recommendations, were excluded from the patient group. The control arm included a group of healthy adults who met the following criteria: no history or symptoms of either bronchial asthma or other pulmonary diseases, no history or symptoms of allergy, no history or symptoms of atopic dermatitis, no history or signs of hypersensitivity to aspirin, negative results of skin tests for 12 common allergens, no first-degree relatives with bronchial asthma or atopic disorders. Spirometry tests were conducted in the Outpatient Clinic according to ERS (European Respiratory Society)/ATS (American Thoracic Society) standards, and allergological tests according to EAACI (European Academy of Allergy and Clinical Immunology) guidelines [12, 19,20,21, 24,25,26].

Note: The authors of the article for this scientific project used their own patient database containing detailed statistical and genetic characteristics used in previous publications. However, this manuscript does not present results published earlier in any other study (the article contains original results not published elsewhere).

Six hundred fifty-two subjects were included in the study. Of this number, there were 345 asthmatic patients at the average age of 48.6 years and 307 healthy subjects at 46.0 years. Detailed patients’ characteristics were presented in Table 2.

The whole group of patients participating in the study was stratified and subjected to statistical analysis. The genotyping was performed by two investigators who were unaware of the phenotypes.

Venous blood samples were collected from the participants onto EDTAK3, and DNA was obtained from peripheral blood leukocyte fraction. The genetic material was isolated using QIAamp DNA Blood Mini Kit (QIAGEN Inc.) according to guidelines provided by the manufacturer (12,13,17,18,19).

An analysis of polymorphisms localized in TGF-β1, TGF-β2 and TGF-β3 genes was conducted on 652 DNA samples with the application of the MassARRAY® system (Bionanopark, Lodz, Poland), using the mass spectrometry technique (MALDI TOF MS – matrix-assisted laser desorption ionization–time-of-flight mass spectrometry), dedicated to nucleic acids.

The authors measured the output level of genomic human DNA and diluted DNA to obtain final cDNA levels = 15 [ng/μl]. In samples with a DNA level lower than 15 [ng/μ], a sufficient undiluted amount of sample was used for analytical purposes. The absorbance ratio A260/A280, identified as a degree of DNA impurities, ranged from 1.7 to 2.0. The DNA levels were measured in all the studied samples.

The first stage of laboratory tests involved performing multiplex-PCR, specific for particular loci, in which 20 fragments of DNA, around 100 bp (base pairs) long, were amplificated. Each DNA fragment had a polymorphic locus in its sequences.

After performing PCR, the remaining unbound dNTPs were dephosphorylated using Shrimp Alkaline Phosphatase (SAP).

The next stage involved performing iPLEX reaction. On the matrix which was formed during the multiplex-PCR reaction, terminal dideoxynucleotides (ddNTPs), corresponding to the allele variant present in the gene, were bonded to MassEXTEND primers with modified mass and complementary to the sequence, located above the polymorphic locus.

After performing the iPLEX reaction, 15 mg resin was placed on each well of a 96-well plate to bind metal ions, such as Na+, K+, Mg2+, which might cause high background interferences in mass spectra. After centrifugation of the plate (5 min., 4000 rpm), the analyzed material was transferred to SpectroCHIP using Nanodispenser RS1000. Its volume ranged from 3 to 25 nl and bonded with the agent filling each of the 96 wells on the chip.

The first stage of the mass spectrometer analysis included matrix laser desorption, needed for DNA analysis. SpectroCHIP is placed inside the spectrometer, and each place with a DNA sample is ionized with a laser in the vacuum. An energy beam used in laser ionization must be well adjusted so that the particles do not get fragmented but bounced only. Next, the particles proceed to a time-of-flight analyzer. The particles going to the analyzer are accelerated with an electric impulse and start drifting through the analyzer chamber. At the end of the analyzer, an ion detector is connected with a device that registers the time between the acceleration impulse and the moment the particle hits the sensor. A result of the mass-to-charge ratio value corresponds to a value we obtain if we follow a low of physics saying that for an impulse of particular amperage, the time-of-flight of ions gets longer if their molecular weight increases. An analysis of the sample using the MassARRAY mass spectrometer enabled us to obtain raw results presented in the form of mass spectra. The mass spectra, in turn, allowed the identification of polymorphisms in the following studied genes: TGF-β1, TGF-β2, and TGF-β3. The graphs could enable us to determine the reaction yield and probability of the obtained results.

The Kruskal-Wallis rank-sum test was applied to compare asthma control test scores in genotypes, whereas the two-sample Wilcoxon test was used to compare the scores in minor allele carriers (MAC). Due to a large number of comparisons, the p-values were adjusted using the Benjamini & Hochberg procedure. Treating the ACT score as continuous, linear regression models were developed for both genotypes and MAC statuses to assess the influence of variables on the ACT score. The backward stepwise selection was based on the Akaike Information Criterion for a fitted parametric model. The overall performance of the models was assessed utilizing multiple R2 and RMSE. At the same time, the influence of certain factors required a calculation of standardized beta coefficients after estimating them using standard deviation. Although measured on a continuous scale, the response variable (ACT score) can be treated as an ordinal variable. Below I check whether the application of ordinal regression (i.e., proportional odds logistic regression) would bring about any changes. The logistic regression was designed in the same manner to test whether the genotypes or MAC statuses will allow predicting reasonable asthma control (ACT≥25). The analysis was performed in STATISTICA 13.1 (Dell, USA) and R programming language (packages: rms, cutopointr, caret).