Cystic fibrosis (CF) is a multi-organ, autosomal recessive disease caused by mutations in a gene located on the long arm of chromosome 7, which encodes the cystic fibrosis transmembrane conductance regulator (CFTR). The absence or reduced function of the CFTR protein is associated with multi-organ dysfunction and a shortened lifespan.^[1–3]

CFTR mutations affect epithelial ions and water transport in the cells of the respiratory system, gastrointestinal tract, hepatobiliary system, reproductive organs, and sweat glands. Over 2,000 mutations in the CFTR gene have been identified, with the most commonly detected being the deletion of phenylalanine at position 508 (delF508).^[3]

In the Bulgarian population, a total of 38 mutations have been identified. Besides delF508, which accounts for 83%, only four others have a frequency above 2% - N1303K, G542X, 2183AA-G, and R347P. A combination of rare genetic defects has been found in only 16% of patients.^[4,5] The diversity of genotypic variations leads to a wide range of clinical manifestations and disease severity across different age groups.^[6]

Pulmonary involvement is the primary cause of morbidity and mortality in CF patients, but extrapulmonary manifestations also negatively impact disease progression. Early diagnosis is crucial for increasing survival rates through timely treatment and monitoring by a multidisciplinary team in CF treatment centers.^[7]

The present study aimed to conduct a retrospective analysis of confirmed genetic variants, their associated clinical manifestations at diagnosis, disease progression, and mortality rates in cystic fibrosis patients in the Pleven region over a 28-year period.

The study analyzed 77 patients aged 1 to 32 years with cystic fibrosis diagnosed and treated at the Pediatric Disease Clinic in Pleven from 1989 to 2017. The diagnosis was based on clinical symptoms, elevated sweat chloride levels above 60 mmol/L, and DNA analysis. Data were statistically processed using SPSS.

Among the analyzed cohort, sweat test values ranged from 80 to 215 mmol/L, with an average of 105.36 mmol/L. Based on DNA analysis, 71% of patients were heterozygous for delF508, 20% were homozygous for delF508, and 9% had rare or unidentified mutations (N1303K, R347P, or Q220X).

By ethnic distribution, the delF508 mutation was found in 50% of patients of Bulgarian origin, 22% in Turkish families, and 82% in Roma patients.

In 72.6% of cases, the diagnosis was established based on bronchopulmonary symptoms and exocrine insufficiency within the first year of life. Among these, 13.7% were diagnosed before one month of age, 5.2% before six months, and 53.7% between six and twelve months. In 27.4% of patients, the diagnosis was delayed despite early respiratory and gastrointestinal symptoms - 11% were diagnosed after one year, 11% after four years, and 5.4% after nine years.

Among late-diagnosed patients, gastrointestinal manifestations were often misdiagnosed as celiac disease, and in 1.3% of cases, isolated bronchiectasis was observed.

Within the observed cohort, 46.5% had mixed pulmonary and gastrointestinal manifestations, 25.9% had only pulmonary symptoms, 15.4% had malabsorption syndrome with steatorrhea, 9.5% had meconium ileus, 8.2% had an edematous-anemic form, 1.3% had neonatal cholestasis, 2.7% had hepatobiliary involvement in adolescence with the development of portal hypertension and esophageal varices, and 1.3% had cystic fibrosis-related diabetes (Fig. 1).

Pulmonary changes, either isolated or in combination with extrapulmonary manifestations, were present in 72.4% of cases. Early respiratory symptoms appeared in 60% of children by six months, 80% by the end of the first year, and 91% by two years of age. In only 2% of cases, the first disease symptoms appeared after two years of age.

In two observed families, both children were affected but exhibited different early clinical manifestations despite having the same mutation – one with meconium ileus and an edematous-anemic form, and the other with neonatal cholestasis and pulmonary involvement.

Among the children with pulmonary involvement, 73.9% had a primary symptom of dry, non-productive cough. Radiological changes were associated with pneumonia in 30% of cases, emphysema in 10%, atelectasis in 10%, and bronchiectasis confirmed by computed tomography in 19% of cases during follow-up.

Mortality rates in CF patients occurred in 44.1% of cases by the age of one year, 7.8% between one and four years, and 3.9% between four and eighteen years (Fig. 2).

Among patients of Roma origin, mortality occurs in 75% of cases before the age of 2 years. The high lethality at an early age can be explained by the high frequency of homozygous carriage of the delF508 mutation, associated with consanguineous marriages within the Roma population. All children with meconium ileus underwent surgical intervention, but survival was observed in only 2.6% of cases.

Prenatal diagnosis was performed in 22% of families with children diagnosed with cystic fibrosis. The results show that 81% of prenatally examined fetuses were healthy, 6% were affected by the disease, and 12% of pregnancies ended in spontaneous abortion.

A study conducted among 742 patients identified 76 different mutations, with the most common being ΔF508 in 71.3% of patients, followed by G551D (2.9%), G542X (2.3%), 621+1G→T (1.2%), and W1282X (1.2%).^[8]

In Bulgaria, the delF508 mutation is present in 65% of cystic fibrosis patients^[4], detected in 83% in heterozygous and 40% in homozygous states. These findings align with data reported for Germany, Austria, and Slovakia. The high number of rare and unidentified mutations is likely due to migratory processes.^[9]

Analyzing ethnic backgrounds, delF508 was identified in 100% of Roma patients, 46.15% of Turkish-origin patients, and 38% of Bulgarian patients. Since only one molecular defect has been described in the Roma population, this allows for an effective preventive screening program requiring minimal testing.^[4] Among the Turkish population, delF508 is unstable, with high frequencies of R347H, G542X, and Q220X, justifying their inclusion in genetic analyses.^[4,5,9] In Bulgarian patients, the second most common mutation is N1303K, characteristic of Southern Europe. Other mutations such as R1070Q and 1677delTA have been registered in the Black Sea region and are not found in other European countries.^[9,10] The amount of functional CFTR gene correlates with clinical status.^[3]

Several studies aim to establish the relationship between genotype and phenotype in cystic fibrosis patients. The phenotypic expression of the disease is influenced by multiple factors, including timely diagnosis and treatment, social environment, and cultural conditions. Thus, patients with the same genotype, even within the same family, may exhibit significant differences in disease progression.^[11]

Similar to our findings, Santos et al.^[3] analyzed clinical phenotypes of 77 children and adolescents with cystic fibrosis. The most common pathology was pulmonary involvement (92.2%), with pancreatic insufficiency observed in 87% of patients, of whom only 1.3% had pancreatitis. Gastrointestinal symptoms occurred in 46% of cases, with constipation being the most common (33.8%). Hepatobiliary involvement was seen in 62.3%, meconium ileus in 9.3%, neonatal cholestasis in 11.7%, portal hypertension in 6.5%, esophageal varices in 3.9%, and fibrosing colonopathy in 1.3%.^[3]

Numerous studies categorize clinical manifestations according to age. A 40-year study in Toronto observed variations in disease progression in adult-diagnosed cystic fibrosis patients. Among 73 analyzed cases, 39% presented with pulmonary symptoms, 22% had mixed pulmonary and gastrointestinal symptoms, 26% experienced infertility, 9% had diabetes mellitus, and 2% had biliary cirrhosis. Pancreatic insufficiency was found in 73% of adults but only 13% of pediatric cases.^[12]

In neonatal and early infancy, the most frequent phenotypic manifestations include meconium ileus, neonatal cholestasis, edema, anemia, malnutrition, steatorrhea, malabsorption syndrome, inadequate weight gain, and upper dyspeptic syndrome.^[13] Intestinal malabsorption is an early sign, manifesting in 79% of patients by six months and 92% by one year of age.^[14]

During early childhood, patients primarily present with recurrent or chronic cough with bronchial obstruction, non-responsive to treatment, recurrent and chronic pneumonia, growth retardation, chronic diarrhea, rectal prolapse, and hyponatremia with hypochloremia.^[13]

Ong et al.^[15] reported that 85.5% of cystic fibrosis patients in the USA carried the delF508 mutation, with clinical symptoms typically beginning in early childhood with steatorrhea, poor weight gain, and respiratory symptoms.^[15]

In preschool-aged children, chronic cough with purulent expectoration, recurrent bronchial obstruction, nasal polyposis, recurrent abdominal pain, chronic diarrhea, intussusception, failure to thrive, hypertrophic osteopathy, hepatomegaly, chronic hyponatremia, and hypochloremia are observed. In school-aged children, Pseudomonas aeruginosa^[13,16] is isolated from bronchial secretions, and with age, chronic respiratory bacterial infections contribute to loss of lung function and bronchiectasis development.^[15]

Pancreatitis, distal intestinal obstruction syndrome, liver cirrhosis, portal hypertension, and reproductive failure are documented in adolescents and adults with cystic fibrosis.^[13,16] Literature data indicate that rarer symptoms across all ages include meconium ileus, distal intestinal obstruction syndrome, and liver damage^[17], although only 3.9% of patients lack extrapulmonary manifestations.^[18]

Hepatobiliary damage occurs in 10%–15% of patients, peaking in pre-adolescence, with 20%–40% developing clinically significant multilobular cirrhosis, portal hypertension, varices, and bleeding^[16,18] Liver failure is the second leading cause of mortality after pulmonary complications.^[19] In our study, it was responsible for lethality in 2.6% of adolescent cystic fibrosis patients.

Fibrosing colonopathy is a rare condition in cystic fibrosis patients, characterized histologically by submucosal fibrosis with thickening of the muscularis propria, mucosal inflammation, spindle-shaped stenosis, and subsequent stricture. Some authors associate its etiology with excessive pancreatic enzyme intake exceeding 10,000 IU lipase/kg/day.^[20] Among our observed patients, only one child developed fibrosing colonopathy after undergoing surgery for a mucinous cystadenoma of the right ovary, complicating the differentiation of abdominal pain.

The diversity in phenotypic expression and the absence of neonatal screening in Bulgaria contribute to late diagnosis in 27.4% of our observed cases. Given the availability of modern therapies, attention must be directed toward early diagnosis to improve survival and quality of life.

Globally, there has been progress in increasing the average life expectancy of cystic fibrosis patients - from 30 years in 1999 to 36.3 years in 2006, and currently, according to the European Cystic Fibrosis Registry, it is 51.7 years. Europe reports the lowest mortality rates in the 0-4 age group, with higher rates in adolescence.^[21]

A study in Iran involving 105 pediatric cystic fibrosis patients observed the highest lethality in the 2-6-year age range (42.9% of cases).^[22] These results align with our analysis, showing the highest mortality rates before age two, particularly among the Roma ethnic group. This may be explained by the higher frequency of delF508 homozygotes and the manifestation of meconium ileus and edematous-anemic forms of the disease.

Additionally, 13.3% of Iranian patients had siblings with cystic fibrosis, compared to only 2.6% in our study, likely due to prenatal diagnosis in the observed cohort.^[22]

The analysis indicates that the most common mutation among cystic fibrosis patients is heterozygous delF508. The predominant clinical manifestations at diagnosis and during follow-up include the bronchopulmonary form of the disease, followed by gastrointestinal and hepatobiliary involvement. The significant proportion of delF508 homozygotes with phenotypic manifestations of meconium ileus and edematous-anemic forms of the disease contribute to high mortality rates in children under one year, regardless of ethnicity.

A multidisciplinary approach delays disease progression. However, due to significant variations in severity and affected organs, cystic fibrosis remains a challenge in clinical practice. Implementing nationwide neonatal screening in Bulgaria will enable earlier diagnosis, reducing mortality and improving life expectancy and quality of life.

The authors have no funding to report.

The authors have declared that no competing interests exist.

Nadia Kolarova: data analysis and interpretation; Evgeni Mekov: data collection; Nikolay Yanev: drafting the article; Vania Nedkova: conception and design of the work.