Methods
Sweat chloride levels were tested according to guidelines [
26]. We screened for a panel of mutations and for the most common
CFTR rearrangements [
27]; thus, we carried out gene sequencing (detection rate 95%) [
28,
29] in patients in which one or both mutations resulted undetected after first level analysis [
30]. We analyzed 7 intragenic
CFTR short tandem repeats [
31] to verify that both members of four sibling-pairs carrying only one known mutation had the same
CFTR genotype. Molecular analysis revealed more than 50 different
CFTR mutations in our patients and only for a half of them functional studies had defined the molecular effect and the class; furthermore, some of these latter mutations may combine more defects and can be attributed to more classes [
2]. Thus, we did not perform correlations between the
CFTR genotype and clinical parameters. However, Additional file
1: Table S1 provides the
CFTR genotype of each patient included in the study.
Meconium ileus was defined using the criteria described [
15]. The forced expiratory volume (FEV
1), expressed as the percentage of predicted value for age, according to standardized reference equations for spirometry [
32] was recorded. To avoid age-related differences between siblings of each pair, we recorded as current FEV
1 the last value of the younger sibling and that of the older sibling at the same age. In the case of patients who had died or had undergone lung transplantation we recorded the last FEV
1 and the value of the living or non-transplanted member of the sibling-pair at the same age. Given the inter-individual variability of FEV
1 and the evolution of lung damage with age [
33] the patients were classified as severe or mild according with the criteria by Schluchter et al., that take into account both the FEV
1 value and age [
34,
35]. The airway colonization by
P. aeruginosa was identified by sputum or oropharyngeal swab culture. Chronic infection was defined according to the modified Leeds criteria [
36].
Fecal pancreatic elastase was evaluated annually and at least 3 months before enrollment. Pancreatic sufficiency was defined on the basis of fecal pancreatic elastase-1 higher than 200 mcg/g measured in the absence of acute pancreatitis or gastrointestinal diseases. Pancreatitis was defined as acute or chronic according to the report from International study group of pediatric pancreatitis [
37] excluding all known causes of pancreatitis. Liver disease was evaluated by means of clinical, biochemical or ultrasonography abnormalities recorded in two consecutive examinations within a 3-month period, in the absence of other causes of congenital or acquired chronic liver disease [
38]. Patients were considered as affected by CFLD when they had liver cirrhosis, considered as the extreme phenotype to define liver disease in our study, with imaging techniques showing nodular hepatic parenchyma and signs of portal hypertension [
17]. A glucose tolerance test was performed annually in all patients with CF and the diagnosis of CFRD was made according to the standard American Diabetes Association criteria [
39]. Finally, the history of nasal polyposis requiring surgery was evaluated.