Every CFTR variant counts - Target-capture based next-generation-sequencing for molecular diagnosis in the German CF Registry.

BACKGROUND: In times of genotype guided therapy options, a total of 3.2 % of people with CF (pwCF) in the German CF Registry[1] only have one or no CFTR-variant detected after genetic analysis. Additionally, genetic data in the Registry can be documented as free text and can therefore be prone to error. In order to allow the greatest possible amount of pwCF access to modern therapies, we conducted a re-evaluation of free text entries and established a custom-whole-CFTR-locus NGS-approach for all pwCF who remained without genetic confirmation afterwards. METHODS: To this end, we assembled 731 free text variants of 655 pwCF in the German CF Registry. All variants were evaluated using ClinVar, HGMD and CFTR1/2, corrected in the Registries' database and uploaded to ClinVar. PwCF whose diagnosis remained uncertain as well as additional pwCF or pwCFTR-RD that were assembled through a nationwide call for testing of unclear cases were offered genetic analysis. Samples were analysed using a target-capture based NGS-custom-design-panel covering the entire CFTR-locus. RESULTS: Evaluation of free text variants led to the discovery of 43 variants not formerly reported in the context of CF. The Registries' dropdown list was extended by 497 variants and over 500 pwCF were provided with their most up-to-date genotype. Samples of 47 pwCF/pwCFTR-RD were sequenced via NGS with an overall success rate of 61.7 %, resulting in implementation of entire CFTR-genotyping into routine diagnostics. CONCLUSION: Entire CFTR-genotyping can greatly increase the genetic diagnostic rate of pwCF/pwCFTR-RD and should be considered after inconspicuous CFTR screening panels in CFTR-diagnostics.

Elexacaftor - Tezacaftor - Ivacaftor treatment improves systemic infection parameters and Pseudomonas aeruginosa colonization rate in patients with cystic fibrosis a monocentric observational study.

Background & aims: The CFTR-modulating therapy Elexaftor - Tezacaftor - Ivacaftor (ETI) has been widely prescribed since its approval in 2020 in the European Union. The aim of this study was to methodically evaluate the effects of an ETI treatment on clinical, biochemical data and Pseudomonas colonization in order to demonstrate its efficacy. Methods: This prospective monocentric study comprised 69 patients diagnosed with cystic fibrosis aged at least 12 years and treated with ETI between September 2020 and November 2021. Clinical and laboratory data of each patient and study visit were collected before and after 24 weeks of ETI treatment. Follow-up status of Pseudomonas aeruginosa (PsA) colonization was assessed after one year of therapy by regularly determined sputum or throat swab samples. Results: Marked improvements biochemical markers of systemic inflammation as white blood cell count, levels of immunoglobulins A, G and M and albumin within 24 weeks of therapy were observed. ETI treatment proved to be effective as seen by amelioration of lung function and sweat chloride concentration. Assessment of PsA colonization status revealed a conversion from a positive to negative detection in 36% of the cases after one year of therapy. Conclusions: ETI treatment effectively improves systemic inflammation parameters and shows promising results in PsA status conversion.

Increase of liver stiffness and altered bile acid metabolism after triple CFTR modulator initiation in children and young adults with cystic fibrosis.

BACKGROUND: Novel cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies (elexacaftor/tezacaftor/ivacaftor-ETI) promise clinically significant and sustained improvements for patients with cystic fibrosis (CF). In this study, we investigated the impact of ETI therapy on liver stiffness and bile acid metabolism in a cohort of children and young adults with CF. METHODS: A prospective observational study (NCT05576324) was conducted from September 2020 to November 2021 enrolling CF patients naive to ETI. Standard laboratory chemistry, sweat test, lung function, share wave velocity (SWV) derived by acoustic radiation force impulse imaging (ARFI) and serum bile acid profiles were assessed before and 6 months after induction of ETI therapy. RESULTS: A total of 20 patients (10 aged <20 years) completed the study. While lung function and BMI improved after ETI therapy, ARFI SWV increased in CF patients <20 years of age (from 1.27 to 1.43 m/s, p = 0.023). Bile acid (BA) profiles revealed a decrease in unconjugated (5.75 vs 1.46, p = 0.007) and increase in glycine-conjugated derivatives (GCDCA) (4.79 vs 6.64 p = 0.016). There was a positive correlation between ARFI SWV values and GCDCA (r = 0.80, p < 0.0001). Glycine-conjugated BA provided high diagnostic accuracy to predict increased ARFI measurements (AUC 0.90) and clinical (Colombo) CFLD grading (AUC 0.97). CONCLUSIONS: ARFI SWV and bile acid profiles provide evidence for early increase in liver stiffness and altered bile acid metabolism in young CF patients after initiation of ETI and may serve as synergistic measures for detection of hepatic complications during ETI therapy.

Infliximab therapy of relapsing tracheal stenosis in a pediatric patient with granulomatosis with polyangiitis: a case report.

BACKGROUND: Granulomatosis with polyangiitis is a granulomatous, necrotizing small-vessel vasculitis affecting both children and adults. However, subglottic tracheal stenosis appears more frequently in the pediatric cohort. To date, granulomatosis with polyangiitis is often treated with steroids, cyclophosphamide, azathioprine, or rituximab, but tumor-necrosis-factor-α-antagonistic drugs are increasingly gaining significance in treatment of refractory cases. CASE PRESENTATION: We report the case of a 15-year-old Caucasian male diagnosed with proteinase-3-positive granulomatosis with polyangiitis with acute shortness of breath. X-ray and magnet resonance imaging showed extensive subglottic narrowing. Forced expiratory volume in 1 s was reduced to 50% of age norm, with massively increased effective airway resistance. The patient initially responded very well to high-dose steroids and maintenance therapy with azathioprine. He was subsequently treated with four doses of rituximab, and levels of proteinase 3 antibodies normalized. After 6 months of clinical remission, the patient presented again with acute respiratory symptoms. Again, he was treated with high-dose steroids, but showed poor clinical response this time. Therefore, we decided to commence a tumor-necrosis-factor-α-antagonistic treatment with infliximab, under which our patient achieved clinical remission and normalization of lung function parameters. CONCLUSIONS: The use of tumor-necrosis-factor-α-antagonistic agents might be a promising alternative for the treatment of refractory tracheal stenosis in pediatric patients with granulomatosis with polyangiitis.

DMBT1 is upregulated in cystic fibrosis, affects ciliary motility, and is reduced by acetylcysteine.

BACKGROUND: Cystic fibrosis (CF) is the most common genetic disorder in the Caucasian population. Despite remarkable improvements in morbidity and mortality during the last decades, the disease still limits survival and reduces quality of life of affected patients. Moreover, CF still represents substantial economic burden for healthcare systems. Inflammation and infection already start in early life and play important roles in pulmonary impairment. The aim of this study is to analyze the potential role of DMBT1, a protein with functions in inflammation, angiogenesis, and epithelial differentiation, in CF. RESULTS: Immunohistochemically DMBT1 protein expression was upregulated in lung tissues of CF patients compared to healthy controls. Additionally, pulmonary expression of Dmbt1 was approximately 6-fold increased in an established transgenic mouse model of CF-like lung disease (ENaC tg) compared to wild-type mice as detected by qRT-PCR. Since acetylcysteine (ACC) has been shown to reduce inflammatory markers in the airways, its potential influence on DMBT1 expression was analyzed. A549 cells stably transfected with an expression plasmid encoding the largest (8kb) DMBT1 variant (DMBT1+ cells) or an empty vector control (DMBT1- cells) and incubated with ACC both showed significantly reduced DMBT1 concentrations in the culture medium (p = 0.0001). To further elucidate the function of DMBT1 in pulmonary airways, respiratory epithelial cells were examined by phase contrast microscopy. Addition of human recombinant DMBT1 resulted in altered cilia motility and irregular beat waves (p < 0.0001) suggesting a potential effect of DMBT1 on airway clearance. CONCLUSIONS: DMBT1 is part of inflammatory processes in CF and may be used as a potential biomarker for CF lung disease and a potential tool to monitor CF progression. Furthermore, DMBT1 has a negative effect on ciliary motility thereby possibly compromising airway clearance. Application of ACC, leading to reduced DMBT1 concentrations, could be a potential therapeutic option for CF patients.

Potential for Radiation Dose Reduction in Dual-Source Computed Tomography of the Lung in the Pediatric and Adolescent Population Compared to Digital Radiography.

Low-dose dual-source computed tomography (DSCT) protocols for the evaluation of lung diseases in children and adolescents are of importance since this age group is particularly prone to radiation damage. The aim of this study was to evaluate image quality of low-dose DSCT of the lung and to assess the potential of radiation dose reduction compared to digital radiographs (DR). Three groups, each consisting of 19 patients, were examined with different DSCT protocols using tin prefiltration (Sn96/64/32 ref. mAs at 100 kV). Different strengths of iterative reconstruction were applied (ADMIRE 2/3/4). DSCT groups were compared to 19 matched patients examined with posterior-anterior DR. Diagnostic confidence, detectability of anatomical structures and small lung lesions were evaluated on a 4-point Likert scale (LS 1 = unacceptable, 4 = fully acceptable; a value ≥ 3 was considered acceptable). Effective dose (ED) was 31-/21-/9-fold higher in Sn96/Sn64/Sn32 compared to DR. Diagnostic confidence was sufficient in Sn96/Sn64 (LS 3.4/3.2), reduced in Sn32 (LS 2.7) and the worst in DR (LS 2.4). In DSCT, detectability of small anatomical structures was always superior to DR (p < 0.05). Mean lesion size ranged from 5.1-7 mm; detectability was acceptable in all DSCT groups (LS 3.0-3.4) and superior to DR (LS 1.9; p < 0.05). Substantial dose lowering in DSCT of the pediatric lung enables acceptable detectability of small lung lesions with a radiation dose being about 10-fold higher compared to DR.

Transfusion-related alloimmune neutropenia with no pulmonary complications: one donor-five cases.

BACKGROUND: Neutrophil alloantibodies are well-known triggers of transfusion-related acute lung injury (TRALI) and also cause immune neutropenia. Alloimmune neutropenia due to transfusion is an isolated phenomenon that is only rarely identified. Its incidence is specified in the literature as being less than one in 10,000 transfused plasma-containing units. We expect that this phenomenon is underreported. STUDY DESIGN AND METHODS: We observed five cases of alloimmune neutropenia with no respiratory complications with only one case initially reported as a suspected transfusion reaction. The other four cases were detected in the course of the subsequent lookback investigation. RESULTS: The first case was reported as a potential transfusion reaction when a female patient showed a decrease in the white blood cell count after a platelet (PLT) transfusion. Examinations of the donor blood revealed an antibody against the human neutrophil antigen HNA-1b; the recipient was typed HNA-1b positive and HNA-1a negative. After examining the blood counts of other patients who previously received PLT concentrates from the same donor, we identified four other patients with an unreported decrease in the leukocyte and/or granulocyte count of more than approximately 50% after transfusion. CONCLUSION: HNA antibodies are generally regarded as potential triggers of TRALI. Here we describe an HNA antibody that reproducibly caused transfusion-related neutropenia only without pulmonary complications. Factors predisposing patients to TRALI development are widely discussed. Our case suggests that antibody characteristics are also relevant in the development of TRALI. Current measures to prevent TRALI should also prevent transfusion-related alloimmune neutropenia.