A total of 49 patients with CF diagnosed by (having shown [chloride] (≥60mEq/L)) the sweat test were included. All participants were homozygous or compound heterozygous for CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) pathogenic variants (class I or II). No patient was diagnosed via the neonatal screening test. The study was approved by the Institutional Ethics Committee of the University of Campinas (#570/2004). All participants or their parents signed a consent form before the beginning of the study.

The DNA was obtained via phenol-chloroform extraction. The [DNA] used for analysis was 50ng/mL, evaluated using a GE NanoVue™ Spectrophotometer (GE Healthcare Biosciences, Pittsburgh, USA).

The polymerase chain reaction (PCR) for amplification of ADIPOQ and STATH genes was performed with bidistilled water, 10× Taq buffer with (NH4)2SO4, MgCl2 (25mM), dNTP (25mM of each nitrogenous base), primers (0.2ρmol), Taq polymerase (5U) and genomic DNA (50ng/mL). The primers used in the analyses are shown in Table 1. The PCR conditions for ADIPOQ and STATH genes were 94°C/5min; 35 cycles of 94°C/1min, annealing temperature/1min and 72°C/2min; and 72°C/7min. The annealing temperature for each fragment analyzed is shown in Table 1.

The sequencing of exons and exon/intron junctions of STATH and ADIPOQ genes was performed in MegaBACE® 1000 (GE Healthcare, Pittsburgh, PA, USA) using the DYEnamic ET Dye Terminator Cycle Sequencing Kit (with Thermo Sequenase™ II DNA Polymerase) (GE Healthcare, Pittsburgh, PA, USA) following the manufacturer’s recommendations. The sequences were analyzed using the Chromas Lite software, version 2.3.3.0 Chromas MFC application.18

The clinical markers included were: CFTR pathogenic variants, age at the time of diagnosis, age at the onset of pulmonary and digestive symptoms, first infection by P. aeruginosa, spirometry, Shwachman-Kulczycki score, Kanga score, and transcutaneous oxygen saturation of hemoglobin.

For age at the time of diagnosis, age at the onset of pulmonary and gastrointestinal symptoms, and time until the first isolation of P. aeruginosa, the following groups were used: up to two months old, 13–36 months old, above 36 months old. In the case of digestive symptoms, meconium ileus was considered as an additional clinical marker. For P. aeruginosa, some patients were decolonized before the beginning of the study and were not included in the descriptive analysis.

Spirometric values were described considering forced expiratory volume in one second (FEV1%) as: (i) severe obstruction: <40%; (ii) moderate obstruction: ≥40% to <60%; (iii) mild obstruction: ≥60% to <80%; (iv) normal lung function: ≥80%.

Spirometry was performed using a speedometer CPFS/D model (Med Graphics, Saint Paul, Minnesota, USA). Data were recorded using the BREEZE PF software Version 3.8 B for Windows 95/98/NT.

The Shwachman-Kulczycki score was classified as excellent (86–100), good (71–85), mild (56–70), moderate (41–55) and severe (40 or less). The score was evaluated by two professionals, because it was a subjective analysis. In case of disparity between evaluators, a third evaluation was performed. The Kanga score was analyzed considering the presence or absence of exacerbation in the points obtained.

The transcutaneous oxygen saturation of hemoglobin was categorized as: (i) normal: ≥95%; (ii) mild hypoxemia: ≥91 to <95; (iii) moderate hypoxemia: ≥85 to <90; (iv) severe hypoxemia <85%.

Of the 49 patients with CF included in the study, 25/49 (51.02%) were female. Ages ranged from one to 26 years old, with the mean age being 9.71±6.06 years old. The mean age at the time of diagnosis was 29.70±2.47 months old. For the first P. aeruginosa, the mean age was 38.11±3.17 months old (Tables 2 and 3).

In relation to the CFTR genotype, there was a high prevalence of homozygous p.Phe508del (63.26%). The p.Phe508del allele was the most prevalent (79.59%), followed by p.Gly542X (9.18%), p.Arg1162X (6.12%), p.Asn1303Lys (3.06%) and p.Arg553X (2.04%) (Table 2).

The three exons of ADIPOQ gene and four of the five exons of STATH were analyzed. Exon 1 of the STATH gene was not analyzed being considered a non-translated region. The three and four exons were analyzed with a single primer pair. No changes (rare genetic variants) were found in all patients with CF for all fragments. Also, there is no need to compare among the homozygous subjects for CFTR and compound heterozygous subjects for CFTR because all CFTR variants included are severe variants.

According to age range, most patients were included in the range between zero and ten years old (69.38%). Due to the inclusion of patients with pathogenic mutations in the class I and II CFTR group, these data may be associated with severe prognosis and low life expectancy considering the presence of severe mutations. However, the survival rates and prognosis of patients with CF have improved. One of the factors that has been associated with this fact is the systematic care of patients in specialized centers.19

Regarding sex, a uniform distribution was observed, a fact associated with autosomal recessive inheritance. However, the literature reports a slight predominance of males compared to females, increasing with the patients’ age.20,21 The lower prevalence in females may occur due to the vulnerability of females to certain clinical characteristics, such as the occurrence of diabetes mellitus.

The mean age at the time of diagnosis (2.47 years old) was higher than that found by Dorfman et al. (2008)22 (0.36 years old) in a group of 611 homozygous p.Phe508del. The average age at the time of the first infection by P. aeruginosa (3.17 years old) was lower than the one reported in the same study (7.5 years old). This difference can be explained by the lower age at the time of diagnosis of the previous study, compared to ours, suggesting that the early treatment of these patients can delay the colonization by P. aeruginosa.

Low adiponectin levels promote inflammation and are associated with increased insulin resistance and high risk of cardiovascular disease.23 Furthermore, adiponectin is associated with the regulation of energy balance, and in CF, chronic energy deficiency and thus higher [adiponectin] levels occur.24

Adiponectin has anti-inflammatory properties, mainly inhibiting the production of pro-inflammatory cytokines and inducting anti-inflammatory factors. However, higher levels of adiponectin were found in patients with CF compared to healthy subjects and as explanation, the presence of deficiency in the energy balance was considered24; moreover, the absence of correlation between inflammation markers [e.g. C-reactive protein (CRP) and fibrinogen] and adiponectin was reported, suggesting that adiponectin levels are not reduced in CF, even in the presence of low-grade inflammation or chronic infection/inflammation.23,25

In addition, functional variants in ADIPOQ have been associated with levels of circulating adiponectin.8,12 Some of these variants result in high levels of adiponectin, which could support a better clinical outcome of patients with CF, since adiponectin acts in suppressing inflammation-related diseases and improving nutritional status.2 In this context, two variants (exon 2 and 3) that decrease the level of circulating adiponectin were described.26 However, in our study, the presence of these variants was not supported.

In our study, it was not possible to establish the relationship between variants in ADIPOQ and CF severity, since no variants were found.

Studies relating STATH and CF are scarce. However, it is known that statherin is an antimicrobial peptide expressed in the upper airways and nasal secretions involved in the development of biofilm in the oral cavity, mediating bacterial adhesion.2 It has recently been identified as the most prominent protein in the saliva’s interaction with atmospheric air.12,14

As a mediator of adhesion of bacteria, statherin has epitopes that promote the growth and adhesion of certain microorganisms in the oral cavity (P. gingivalis) while inhibiting the growth of others (S. aureus),15 although their antibacterial activity against P. aeruginosa has not yet been investigated. It is known that the infection by P. aeruginosa accelerates the decline in lung function.16 Thus, a protein that acts in bacterial adhesion in the oral cavity and upper airways is of extreme interest to studies related to the CF phenotype.

Statherin with high activity levels was found in homozygous p.Phe508del in moderate lung disease.12 This increase in expression was confirmed by an analysis of the mRNA produced by STATH in a sample of 12 patients with CF and moderate and severe pulmonary disease.17

In our study, it was not possible to establish a relation between variants in STATH and CF severity, since no variants were found.