Abstract
Background
Cystic fibrosis (CF) is an autosomal recessive disease caused by variants of CFTR gene. Over 2000 variants have been identified, and new drugs called CFTR modulators have been developed to target specific defects in the CFTR protein. However, these drugs are only suitable for patients with certain variants of CFTR, and eligibility rates vary depending on race and geographical region. This study aimed to reveal the detailed genotype and clinical characteristics of people with CF (pwCF) at our center in Turkey, a developing country, who are not eligible for CFTR modulators.
Methods
A total of 445 pwCF followed up at Marmara University were reviewed retrospectively. Variants of the patients ineligible to CFTR modulators were classified based on American College of Medical Genetics guidelines, CFTR classification, the change in the encoded protein, and the variant type.
Results
The study revealed that 139 (31.2%) patients weren't eligible for CFTR modulators. There were 60 different variants in the 276 alleles, as two were missing. The majority of patients had missense or nonsense variants, and that the most common variant was c.1545_1546del, which can be said unique to this geography.
Conclusion
The study highlights the importance of detecting the variants of ineligible patients in detail to guide future approaches for more targeted and effective interventions in CF care. Testing the effectiveness of CFTR modulators for rare or newly occurring variants is crucial to ensure equal access for pwCF to these therapies from different racial backgrounds and ethnic minorities.
Keywords: CFTR modulator, cystic fibrosis, eligible, genotype, variant
1. INTRODUCTION
Cystic fibrosis (CF; OMIM ♯219700) is an autosomal recessive disease that occurs with a decrease in the function of the CF transmembrane conductance regulator protein owing to variants in the CFTR gene located on chromosome 7. 1 Since its discovery in 1989, more than 2000 variants have been identified in CFTR. 2 Today, CFTR variants are categorized into six classes based on how they affect the function or production of the CFTR protein (Class I–VI). 3 Particularly in the last 20 years, with a further elucidation of its natural history and pathophysiology, CF treatment has managed to go beyond symptom relief and support to intervene in the defective protein. These new drugs, known as CFTR modulators, include potentiators and correctors (ivacaftor, lumacaftor, elexacaftor, and tezacaftor), which are the most used today. 4 , 5 CFTR modulators are small molecules that interact directly with CFTR proteins. 6 First, ivacaftor was approved in the United States in 2012, and the number of countries approving its use has gradually increased. 7 They target specific defects in CFTR protein processing, function, or stability, aiming to restore or enhance CFTR activity.
However, these new, groundbreaking drugs which have been proven to significantly improve patients' lifespan and respiratory functions; are only suitable for people with certain variants in CFTR. 8 Although CF is seen in all races, since CFTR variants vary and cluster according to race and geographical region, the eligibility rate with CFTR modulators reaches 90% in regions where White people are more common, such as the United States and Western/Northern Europe, whereas it has been proven to be lower in some diverse populations and ethnic groups. 9 , 10 This study was initiated to address the considerable genetic variability in Turkey, which arises from its unique position between Europe and Asia, and the observation that certain national groups have fewer variants eligible for existing CFTR modulators, aiming to identify and characterize these differences, potentially paving the way for tailored treatment strategies in these populations. In Turkey, CFTR modulators are still not covered by government reimbursements as of 2024. People with CF with eligible variants file a lawsuit against the social insurance institution through lawyers specialized in this field, and they get the right to use CFTR modulators during the trial.
As the basis of approval for treatment is the variant in CFTR and, accordingly, the level of defects in the CFTR protein, knowing the variants of ineligible patients in detail is essential for guiding future approaches for more targeted and effective interventions. This study was conducted to reveal the detailed genetic and clinical characteristics of people with CF (pwCF) who are being followed up in our clinic, the largest center in Turkey, with variants that are not eligible for CFTR modulators.
2. MATERIALS AND METHODS
2.1. Study population
In this retrospective descriptive study, genetic analysis results of 445 pwCF (301 children [67.7%], 144 adults [32.3%]) registered in European Cystic Fibrosis Society Patient Registry since 2015 and have been following up at Marmara University Selim Çöremen Cystic Fibrosis Center are presented. The patients were diagnosed with CF considering their genetic results (CFTR gene sequence analysis and multiplex ligation‐dependent probe amplification [MLPA]), sweat test results (positive: Cl−> 60 mmol/L, borderline: Cl− = 30–59 mmol/L), and clinical status (recurrent bronchopneumonia, chronic cough and sputum production, bronchiectasis, failure to thrive, meconium ileus, salt depletion, and repeated episodes of pancreatitis).
Patients who were not eligible with any CFTR modulator (ivacaftor, elexacaftor/tezacaftor/ivacaftor and ivacaftor, lumacaftor/ivacaftor, tezacaftor/ivacaftor, and ivacaftor) were included in the study. The eligibility of patients with CFTR modulators was determined through the utilization of the online database provided by Vertex Pharmaceuticals®. Those whose genotypes were eligible but who could not take the drug due to age were excluded.
2.2. Genetic and clinical status
Variant analyses results (CFTR sequencing analysis and MLPA) were obtained from the files and while classifying the variants of the patients, first the variants in both alleles were examined separately and then they were classified according to certain characteristics of the variants.
To determine the characteristics of the variants online databases of Varsome (https://varsome.com/), Franklin Genoox (https://franklin.genoox.com/), Clinvar (https://www.ncbi.nlm.nih.gov/clinvar/), variant list history of CFTR2 (latest version − 07.04.2023) (https://cftr2.org/mutations_history) and CFTR‐France Database (https://cftr.iurc.montp.inserm.fr/cftr) were used. To predict whether the variant affected splicing, Human Splicing Finder (https://hsf.genomnis.com/mutation/analysis) and Splice AI (https://spliceailookup.broadinstitute.org/) were checked. Variants were classified as follows:
-
1.
According to American College of Medical Genetics and Genomics (ACMG), the alleles were divided into groups of benign, likely benign, variant of uncertain significance (VUS), likely pathogenic or pathogenic. 11 By referring to the latest version of the variant list history from CFTR2, the variants were segregated into categories as CF‐causing (CF‐C), varying clinical consequence (VCC), and VUS. Interpretations of variants not found in CFTR2 were checked from the CFTR‐France Database.
-
2.
Missense, nonsense, and frameshift variants were determined according to the change in the encoded protein and the mechanism that caused it. 12 Based on these results, CFTR classification was also performed (Class I variants result in a lack of CFTR protein synthesis. Class II variants lead to altered protein maturation. Class III variants cause defective channel regulation. Class IV variants result in decreased channel conductance. Class V variants reduce CFTR protein synthesis. Class VI variants lead to decreased CFTR stability.). 3
-
3.
Alleles were categorized based on variant type, distinguishing between large exon deletions, exonic and intronic.
-
4.
Point variants were identified and recorded by subtype including base substitution, deletion, deletion‐insertion, duplication, and insertion.
-
5.
Splice site variants were also specified separately for each allele.
The growth values (body mass index [BMI] Z scores at the last examination), maximum forced expiratory volume in the first second (FEV1) value (as a percentage) of the last 12 months, pancreatic insufficiency (PI) status, presence of CF‐related diabetes mellitus (CFRD), colonization of Pseudomonas aeruginosa (PsA), and methicillin‐resistant Staphylococcus aureus (MRSA), number of acute exacerbations and hospitalization days in the last year were recorded. People with fecal elastase value below 200 μg/g were considered as PI and a diagnosis of CFRD was made through an oral glucose tolerance test.
2.3. Statistical analyses
All statistical analyses were performed using IBM SPSS 21.0 program (SPSS Inc.). Chi‐square test was used to compare categorical variables between groups. The normally distributed continuous variables were compared by used the student's T‐test and those without normal distribution by Mann–Whitney U test. The distribution of continuous variables was assessed via The Kolmogorov–Smirnov test and/or Shapiro–Wilk test. While categorical variables were reported as numbers (percentages), continuous variables were given as mean ± standard deviation or median (minimum–maximum) according to their distribution. The statistical significance was defined as p value < .05.
2.4. Ethics
According to the Genetic Diseases Diagnosis Center workflow, information about testing was provided and written patient/parent‐informed content for data collection was obtained from the patients. The study protocol was approved by the ethical committee of Marmara University School of Medicine (Ethical approval number: 04.2024.353).
3. RESULTS
3.1. Demographic and clinical features
From a total of 139 patients (102 children [73.3%], 37 adults [26.7%]), 69 were male (49.6%). Seven (5%) of the patients were from other countries: Iran (1, 7%), Palestine (1, 7%), Russia (1, 7%), and Syria (4, 2.8%). The mean age of the patients at diagnosis was 17.8 ± 41.5 months. The mean age at the time of the study was 12 ± 7.5 years. Colonization of PsA and MRSA was detected in 25 (17.9%) and 5 (3.5%) patients, respectively. One hundred eighteen (84.8%) patients suffered pancreatic insufficiency. The median of the best FEV1 in the last year was 82% (22.9%–110.9%). Thirteen (9.3%) patients were found to have CFRD and all were receiving insulin therapy. The median numbers of acute exacerbations and hospitalizations in the last year were 2 (0–10) and 0 (0–10), respectively. Table 1 compares the demographic data of modulator eligible and noneligible patients.
Table 1.
Clinical course of patients according to modulator eligibility status.
| Patients eligible for modulators median (minimum–maximum) or n (%) | Patients not eligible for modulators median (minimum–maximum) or n (%) | p‐Value | |
|---|---|---|---|
| Age (years) | 13 (1–50) | 11 (1–34) | .10 |
| Gender (female) | 144 (47) | 70 (50.3) | .51 |
| BMIa | −0.15 (−5.86 to 3.93) | −3.6 (−6.26 to 1.75) | .02 |
| FEV1 b (%) | 86.4 (19.9–129.8) | 82 (22.9–110.9) | .12 |
| Number of hospitalizations | 0 (0–6) | 0 (0–10) | .11 |
| Number of exacerbations | 2 (0–10) | 2 (0–8) | .004 |
| Pseudomonas aeruginosa colonization | 67 (21.8) | 25 (17.9) | .33 |
| MRSA colonization | 3 (0.9) | 5 (3.5) | .04 |
| Pancreatic insufficiency | 206 (67.3) | 118 (84.8) | .001 |
| CFRD | 18 (5.8) | 13 (9.3) | .18 |
Abbreviations: BMI, body mass index; CFRD, cystic fibrosis‐related diabetes mellitus; FEV1, forced expiratory volume in the first second; MRSA, methicillin‐resistant Staphylococcus aureus.
At the last examination (Z score).
Best of the last year.
Twelve (8.6%) patients were dead by the time of the study (Table 2).
Table 2.
Genetic and clinical characteristics of patients who were dead during the study.
| Sex | Year of death | Age of death | CFTR variant analysis | FEV1 (%)a | BMIb | Pancreatic insufficiency | Pseudomonas colonization | |||
|---|---|---|---|---|---|---|---|---|---|---|
| 1st allel | 2nd allel | |||||||||
| c.DNA position | Legacy name | c.DNA position | Legacy name | |||||||
| m | 2017 | 14 y | c.1057C>T | Q353X | c.1057C>T | Q353X | 28.1 | −2.67 | + | + |
| f | 2017 | 19 y | delExon 19‐21 | ‐ | delExon19‐21 | ‐ | 32.5 | −0.89 | + | + |
| m | 2017 | 12 y | c.1624G>T | G542X | c.174_177del | D58Efs*32 | 63.1 | −1.22 | + | − |
| f | 2018 | 21 y | c.3909C>G | N1303K | delExon2 | 56.1 | −1.6 | + | + | |
| f | 2019 | 11 y | c.1642_1643del | L548Efs*19 | c.1642_1643del | L548Efs*19 | 58.1 | −1.88 | + | + |
| f | 2020 | 7 y | c.2195T>G | L732X | c.2195T>G | L732X | N/A | −2.89 | + | − |
| f | 2021 | 19 y | c.174_177del | D58Efs*32 | c.174_177del | D58Efs*32 | 38.9 | −2.93 | + | − |
| f | 2021 | 16 y | c.865_869del | R289Nfs*17 | c.865_869del | R289Nfs*17 | 24.1 | −6.26 | + | + |
| m | 2021 | 20 y | c.3909C>G | N1303K | c.1393‐1G>A | ‐ | 47.4 | −2.18 | + | − |
| f | 2022 | 1 y | delExon12‐18 | ‐ | delExon12‐18 | ‐ | N/A | N/A | − | − |
| m | 2023 | 9 y | c.2998del | I1000Lfs*2 | c.2998del | I1000Lfs*2 | 45.7 | −2.53 | + | + |
| f | 2023 | 23 y | c.3472C>T | R1158X | c.1624G>T | G542X | 22.9 | −2.73 | − | + |
Abbreviations: BMI, body mass index; f, female; FEV1, forced expiratory volume in the first second; m, male; y, years.
Best of the last year.
At the last examination (Z score).
3.2. Genotypes
Among the 445 pwCF examined, it was determined that 139 of them (31.2%) did not have variants eligible for current CFTR modulators. These 139 patients had 60 different types of variants in a total of 276 alleles (Table 3). Among the patients, 93 (66.9%) had at least one allele with a Class I variant. Of the seven patients carrying unclassified variants in both alleles, two were PI and five were PS. Of the five patients with an unclassified variant in a single allele, two were PI and three were PS.
Table 3.
Representation of the variants detected in patient cohort.
| c.DNA position | n | Legacy name | CFTR2 |
|---|---|---|---|
| c.1545_1546del | 40 | Y515X | CF‐C |
| c.3909C>G | 25 | N1303K | CF‐C |
| c.2051_2052delinsG | 16 | K684Sfs* 38 | CF‐C |
| c.2998del | 13 | I1000Lfs* 2 | CF‐C |
| c.3964‐3C>G | 13 | ‐ | CF‐C* |
| c.1624G>T | 12 | G542X | CF‐C |
| c.1116+1G>A | 8 | 1248+1G‐>A | CF‐C |
| c.3846G>A | 8 | W1282X | CF‐C |
| c.489+1G>T | 7 | 621+1G‐>T | CF‐C |
| c.3472C>T | 6 | R1158X | CF‐C |
| c.1393‐1G>A | 5 | 1525‐1G‐>A | CF‐C |
| c.3700A>G | 5 | I1234V | CF‐C |
| c.2989‐1G>A | 4 | 3121‐1G‐>A | CF‐C |
| c.2195T>G | 4 | L732X | CF‐C |
| c.3904A>T | 4 | K1302X | CF‐C |
| c.1057C>T | 4 | Q353X | CF‐C |
| c.4277C>T | 4 | S1426F | VUS* |
| c.174_177del | 3 | D58Efs* 32 | CF‐C |
| c.2988+1G>A | 3 | 3120+1G‐>A | CF‐C |
| c.1642_1643del | 2 | L548Efs* 19 | CF‐C |
| c.1911del | 2 | Q637Hfs* 26 | CF‐C |
| c.2502del | 2 | F834Lfs* 10 | CF‐C |
| c.2909‐15T>G | 2 | 3041‐15T‐>G | VCC* |
| c.4251del | 2 | E1418Rfs* 14 | CF‐C |
| c.325_327delinsG | 2 | Y109Gfs* 4 | CF‐C |
| c.1585‐1G>A | 2 | 1717‐1G‐>A | CF‐C |
| c.3618del | 2 | G1208Afs* 3 | N/A |
| c.3717+5G>A | 2 | 3849+5G‐>A | CF‐C |
| c.548del | 2 | L183Pfs* 6 | CF‐C |
| c.865_869del | 2 | R289Nfs* 17 | N/A |
| c.3302T>G | 2 | M1101R | CF‐C |
| c.4115C>A | 2 | P1372H | N/A |
| c.3293G>T | 2 | W1098L | N/A |
| c.2738A>G | 2 | Y913C | N/A |
| c.3131A>G | 2 | E1044G | VUS* |
| c.2658‐1G>C | 1 | 2790‐1G‐>C | CF‐C |
| c.2195T>G | 1 | L732X | CF‐C |
| c.2909‐1G>C | 1 | N/A | |
| c.407T>G | 1 | L136R | N/A |
| c.2417A>G | 1 | D806G | VUS* |
| c.4201G>T | 1 | E1401X | N/A |
| c.1766+3A>G | 1 | 1898+3A‐>G | CF‐C |
| c.3266G>A | 1 | W1089X | CF‐C |
| c.692T>G | 1 | I231R | N/A |
| c.2089dup | 1 | R697Kfs* 33 | CF‐C |
| c.2046del | 1 | K684Nfs* 38 | N/A |
| c.3180del | 1 | G1061Dfs* 22 | VUS* |
| c.3469‐2A>G | 1 | 3601‐2A‐>G | CF‐C |
| c.3963+1del | 1 | ‐ | N/A |
| c.531dup | 1 | G178Wfs* 5 | CF‐C |
| c.3038C>T | 1 | P1013L | VUS* |
| c.4231C>T | 1 | Q1411X | CF‐C |
| c.57G>A | 1 | W19X | CF‐C |
| c.1202G>A | 1 | W401X | CF‐C |
| c.1163C>T | 1 | T388M | VUS* |
| Del exon 2 | 24 | ||
| Del exon 4‐11 | 8 | ||
| Del exon 19‐21 | 4 | ||
| Del exon 12‐18 | 3 | ||
| Del exon 18‐20 | 1 |
Abbreviations: CF‐C, cystic fibrosis causing; VCC, varying clinical consequence; VUS, variants of uncertain significance.
Variant couldn't be found in CFTR2, the result is from CFTR‐French database.
According to ACMG, 236 (85.5%) of the alleles were pathogenic, 34 (12.3%) were likely pathogenic, and 6 (2.1%) were VUS. When examined according to CFTR2, CF‐C variants were detected in 236 (85.5%) alleles. When variants not found in CFTR2 were examined from CFTR‐France Database, it was observed that there were 12 (4.3%) alleles classified as CF‐C, 10 as VUS (3.6%), and 2 (0.7%) as VCC. Eleven (18.3%) variants were not included in either database (Table 3).
One hundred and fifteen (82.7%) patients had homozygous variants and 17 (12.2%) had large exon deletions. The most common variant (40 alleles, 16.9%) was c.1545_1546del (1677delTA), which was a deletion, resulting a nonsense alteration (Table 4). Fourteen (10%) patients had two copies of this variant, while twelve (8.6%) patients had one. When the patients were compared in terms of their genotypes according to this variant, it was found that the exacerbation frequency of homozygous patients was significantly higher than the other two groups (p = .002). While all homozygous patients were PI, this rate was 91.7% in heterozygous patients and 83% in patients who did not carry this variant. However, these differences were not statistically significant (Table 5).
Table 4.
Classification of variants in the patient group.
| EXONIC | Base substitution | Nonsense (40) | c.1624G>T (G542X) | c.2195T>G (L732X) | c.57G>A (W19X) | |
| c.3846G>A (W1282X) | c.3266G>A (W1089X) | c.4201G>T (E1401X) | ||||
| c.3904A>T (K1302X) | c.4231C>T (Q1411X) | |||||
| c.3472C>T (R1158X) | c.1202G>A (W401X) | Splicing (7)a | ||||
| Missense (53) | c.3909C>G (N1303K) | c.4115C>A (P1372H) | c.692T>G (I231R) | |||
| c.3700A>G (I1234V) | c.3293G>T (W1098L) | c.407T>G (L136R) | ||||
| c.4277C>T (S1426F) | c.3302T>G (M1101R) | c.2417A>G (D806G) | ||||
| c.2738A>G (Y913C) | c.3038C>T (P1013L) | c.1163C>T (T388M) | ||||
| c.1057C>T (Q353X) | c.3131A>G (E1044G) | Splicing (6)a | ||||
| Deletion | Nonsense (40) | c.1545_1546del (Y515X) | ||||
| Frameshift (32) | c.1642_1643del (L548Efs) | c.548del (L183Pfs) | c.2046del (K684Nfs) | |||
| c.1911del (Q637Hfs) | c.3618del (G1208Afs) | |||||
| c.2502del (F834Lfs) | c.3180del (G1061Dfs) | |||||
| c.2998del (I1000Lfs) | c.865_869del (R289Nfs) | Splicing (20)a | ||||
| c.174_177del (D58Efs) | c.4251del (E1418Rfs) | |||||
| Deletion + Insertion | Frameshift (18) | c.2051_2052delinsG (K684Sfs) | c.325_327delinsG(Y109Gfs) | |||
| Duplication | Frameshift (2) | c.2089dup (R697Kfs) | c.531dup (G178Wfs) | |||
| INTRONIC | Base substitution | Noncoding (49) | c.2909‐15T>G (3041‐15T‐>G) | |||
| c.3964‐3C>G | c.2989‐1G>A(3121‐1G‐>A) | c.2909‐1G>C | Splicing (47) | |||
| c.1116+1G>A(1248+1G‐>A) | c.2988+1G>A(3120+1G>A) | c.1766+3A>G(1898+3A>G) | ||||
| c.489+1G>T(621+1G‐>T) | c.3717+5G>A(3849+5G>A) | c.2658‐1G>C(2790‐1G‐>C) | ||||
| c.1393‐1G>A(1525‐1G‐>A) | c.1585‐1G>A(1717‐1G‐>A) | c.3469‐2A>G(3601‐2A‐>G) | ||||
| Deletion | c.3963+1del |
Exonic splicing variants are predicted by splicing alghoritms.
Table 5.
Comparison of subgroups based on c.1545_1546del (Y515X) genotype.
| Homozygous c.1545_1546del median (min–max) or n (%) | Heterozygous c.1545_1546del median (min–max) or n (%) | Without c.1545_1546del median (min–max) or n (%) 113 | p‐Value | |
|---|---|---|---|---|
| Number of patients | 14 (10) | 12 (8.6) | 113 (81.2) | |
| Age (years) | 14 (2–34) | 17 (7–23) | 10 (1–33) | .11 |
| Gender (female) | 7 (50) | 7 (58.3) | 56 (49.6) | .84 |
| BMIa | −0.45 (−2.05 to 0.97) | −0.75 (v4.00 to 0.17) | −0.33 (−6.26 to 1.75) | .34 |
| FEV1 b (%) | 89.5 (77.0–104.3) | 74.9 (40–101.3) | 81.7 (22.9–110.9) | .10 |
| Number of hospitalizations | 0 (0–4) | 0 (0–3) | 0 (0–10) | .92 |
| Number of exacerbations | 4 (1–8) | 2 (0–5) | 2 (0–8) | .002 |
| Pseudomonas aeruginosa colonization | 1 (7.1) | 2 (16.7) | 22 (19.6) | .51 |
| MRSA colonization | 0 (0) | 0 (0) | 5 (4.8) | .53 |
| Pancreatic insufficiency | 14 (100) | 11 (91.7) | 93 (83) | .19 |
| CFRD | 3 (21.4) | 2 (16.7) | 8 (7.1) | .15 |
Abbreviations: BMI, body mass index; CFRD, cystic fibrosis‐related diabetes mellitus; FEV1, forced expiratory volume in the first second; MRSA, methicillin‐resistant Staphylococcus aureus.
At the last examination (Z score).
Best of the last year.
The most common exon deletion (24 alleles, 60%) was detected as exon 2 deletion. Homozygous exonic variants were observed in 82 (58.9%) patients, while 15 (10.7%) patients presented with homozygous intronic variants. There were intronic variants in a total of 48 (17.3%) alleles. The most frequent intronic variant detected was c.3964‐3C>G (rs397508652), which was identified in 13 (4.7%) alleles.
A total of 78 (28.2%) alleles manifested splice site variants, with variants observed as homozygous in the splice site region in 29 (20.8%) patients. Thirty‐two (41%) of the splice site variants were located on the exon side and 46 (59%) were intronic. The mostly seen splice site variants were c.2998del (I1000Lfs*2), which was a deletion causing a frameshift, and c.3964‐3C>G. which was an intronic variant as mentioned earlier.
The number of the alleles with base substitution, deletion, deletion‐insertion, and duplication were 146 (52.8%), 70 (25.3%), 18 (6.5%), and 2 (0.7%), respectively (Table 2).
Nonsense variants were found in 80 (28.9%), and missense variants were found in 53 (19.2%) alleles. Homozygous nonsense variants were present in 28 (20.1%) patients, while homozygous missense variants were identified in 19 (13.6%) patients. Fifty‐one (18.4%) of the patients carried a nonsense variant in at least one allele. Frameshift variants were identified in 52 (18.8%) alleles, leading to homozygous frameshift variants in 21 (15.1%) patients. The most common nonsense variant was c.1545_1546del, abovementioned. The most common missense and frameshift variants were c.3909C>G (N1303K) and c.2051_2052delAAinsG (K684Sfs*38), respectively (Table 4).
The total number of alleles was 276, as there were two patients with one variant in only one allele. These people were accepted as CF because their medical history and clinical features were compatible. They both had positive sweat test results. One of the patients had a heterozygous exon 19,20,21 deletion, while the other had a heterozygous c.1163C>T (T388M) variant.
We found eight novel variants in 11 alleles, which were c.2909‐1G>C, c.4115C>A, c.865_869del, c.407T>G, c.3618del, c.3180del, c.692T>G, and c.3963+1delG.
There were 7 pwCF from other nations, and their variants were homozygous c.1585‐1G>A (rs76713772), homozygous c.3700A>G (I1234V), homozygous c.3846G>A (W1282X), homozygous c.2989‐1G> A (rs397508470) (two siblings), homozygous c.1911del (Q637Hfs*26) and homozygous exon 4‐11 deletion, respectively.
4. DISCUSSION
In this study, we present the results of the comprehensive genotyping of pwCF who were ineligible for CFTR modulators followed up in our center. To the best of our knowledge, this is the first study conducted based on genetic results in CFTR modulator ineligible patients in Turkey.
There were 139 patients without eligible variants with CFTR modulators, and this group comprised 31.2% of all our follow‐up patients. Çobanoğlu et al. reported that this rate was approximately 75% in their study published from Turkey in 2020, so thanks to encouraging progress in the development of CFTR modulators, the rate of eligible variants in diverse populations has increased significantly. 8 On the other hand, this rate is still much higher than non‐Hispanic white patients from the USA, Canada, or northern/western European countries, where the frequency of the c.1521_1523del (p.Phe508del, F508del) variant is over 70%. 6 , 7 , 9 Yet again, when minorities living in these countries were examined, it was shown that the eligibility rate decreased. 13 While the modulator eligibility of non‐Hispanic whites living in the United States was reported to be 90%, the same rate was 70% for blacks and 75% for Hispanics living in the country. 14 The same situation applies to non‐Scandinavian countries. 10
In their paper published in 2022, Vaidyanathan et al. showed that 48.8% of South Asian patients registered with the UK Registry and 40.6% of patients registered with the Canadian Registry carried variants that were not eligible for CFTR modulators. 15 Yiallouros et al. reported modulator eligibility rates for pwCF as 74.5% in their article published from Cyprus. 16 In another study published from South Africa, where 70% of the patients included in the study were Caucasians, the triple CFTR modulator (elexacaftor/ivacaftor/tezacaftor) eligibility rate was given as 80%. 17
The most common variant in our CFTR modulator ineligible patients was c.1545_1546del (1677delTA), which was ranked 50th according to allele frequency in CFTR2 database (www.cftr2org). Which is a class I variant that involves the loss of two nucleotides in the gene and leads to a frameshift, causing a premature stop codon. In fact, this variant was already reported as the second most common variant in the entire patient group in a comprehensive genotype analysis study conducted in the same cohort, with the information that 100% of the patients with that variant were PI, 30% had chronic PsA colonization and 30% had liver involvement. 1 However, no significant difference was observed in FEV1 (%) values compared to the other four most frequently observed variants. Also, Bonyadi et al. reported this variant as the second most in Azeri Turkish patients lived in Iran, Turkey's neighbor from the south. 18 In an article published in 2019 from the Czech Republic, this variant was stated as the most common variant registered in their registry which was also reported to be common in Black Sea region. 19 In this study, when homozygous and heterozygous pwCF carrying the mentioned variant were compared in terms of clinical features, it was shown that PI rate in the homozygous group was statistically higher, no statistically difference was reported in terms of other clinical courses. Likewise, in our patient group, despite not being statistically significant, 100% of patients carrying this variant were PI. Additionally, the exacerbation frequency of these patients was significantly higher than the others.
Only nine of our patients did not have class I or class II variants. People with CF carrying two variants classified within the first three classes often display severe CF symptoms, primarily characterized by PI. 6 Therefore, the high rate of PI (84.8%) in the non‐eligible patients participating in the study was not surprising.
Some studies have identified alternative therapies for pwCF who are ineligible for CFTR modulators. These include readthrough agents for nonsense variants, nucleic acid‐based therapies (such as RNA or DNA‐based) and cell‐based therapies. 20 In our study, of the 52 (37.4%) patients who carried a nonsense variant in at least one allele, 28 (20.1%) were homozygous. Approximately 10% of the patients in Europe carry nonsense variants (class I) causing a premature stop to the translation of CFTR. 21 Readthrough agents aim to override these premature stop signals. 22 Aside from studies whose outcomes weren't as expected, such as ataluren, there are also promising articles in the literature. 23 , 24 Ribonucleic acid (RNA) editing is another option that has been working on. 25 In principle it has a great potential, but the challenge is to apply it to patients. For that matter, antisense oligonucleotides (ASOs) are promising targeting CFTR. 26 There have been significant advancements in gene therapy, with various clinical trials ongoing trying different methods to insert healthy CFTR gene copies into the cell, as the lung is a difficult target organ with barriers that have evolved for protection, it generally keeps out the gene transfer agents. 27 Despite being promising, the development of these therapies is still in preclinical or early clinical phases.
Undoubtedly, in the context of CFTR modulators, the principle that a drug cannot be presumed eligible with a variant without specific study holds true. Unfortunately the studies and clinical trials leading to the development and approval of the modulators may not fully represent the diversity of the CF population. 9 Many CFTR variants have not been included in major drug trials, primarily because these trials tend to focus on the most common variants that affect the largest segment of the CF population. 28 So rarer variants, which are generally more prevalent in diverse populations, being underpresented in drug trials, impacts modulator eligibility for these groups. 29
This study, while offering comprehensive insight into the genetic profiles of pwCF who are ineligible for CFTR modulators in Turkey, does have some limitations. Our findings are based on patients from a single center, which could limit the generalizability of the results to the wider Turkish CF population. These considerations highlight the importance of continuous genetic research and the potential benefit of multicenter studies to provide a more comprehensive understanding of CFTR variants and their treatment implications.
5. CONCLUSION
Detailed documentation of the genotypes of individuals with genetic diseases is crucial for both patients and society. In the case of CF, knowing the patient's genotype enables personalized treatments and genetic counseling. It is known that not running genetic tests for diseases like CF, is one of the biggest barriers for modulator equity. 10 To date, most drug studies have focused on developing treatments for F508del, which can help around 85% of pwCF. 6 But in the era of CFTR modulators, these promising drugs should not become another branch of health inequality. It is essential to ensure that CFTR modulators are equally accessible to all patients, including those with rare or newly occurring variants. Focusing on studies of ineligible groups, particularly those with class I and deletion variants, should be pivotal for improving treatment options.
AUTHOR CONTRIBUTIONS
Ceren Ayça Yıldız: Formal analysis; software; writing—original draft; investigation; data curation; validation. Merve Selçuk Balcı: Software; data curation; investigation. Şeyda Karabulut: Software; data curation; investigation. Zeynep Münteha Başer: Data curation; validation. Mine Yüksel Kalyoncu: Software; data curation; investigation. Neval Metin Çakar: Software; data curation; investigation. Müge Merve Akkitap Yiğit: Data curation; software. Eda Esra Baysal: Software; data curation. Fulya Özdemircioğlu: Software; data curation. Burcu Uzunoğlu: Software. Gamze Taştan: Software. Pınar Ergenekon: Methodology; writing—review & editing; supervision. Yasemin Gökdemir: Methodology; writing—review & editing; supervision. Ela Erdem Eralp: Methodology; writing—review & editing; supervision. Fazilet Karakoç: Project administration; methodology; conceptualization; writing—review & editing. Pınar Ata: Methodology; conceptualization; writing—review & editing. Bülent Karadağ: Project administration; methodology; conceptualization; writing—review & editing.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.
ETHICS STATEMENT
According to the Genetic Diseases Diagnosis Center workflow, information about testing was provided and written patient/parent‐informed content for data collection was obtained from the patients. The study protocol was approved by the ethical committee of Marmara University School of Medicine.
Yıldız CA, Selçuk Balcı M, Karabulut Ş, et al. Beyond the 10%: Unraveling the genetic diversity in Turkish cystic fibrosis patients not eligible for CFTR modulators. Pediatr Pulmonol. 2024;59:3250‐3259. 10.1002/ppul.27181
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.