With the introduction of elexacaftor/tezacaftor/ivacaftor (ETI), there has been significant impact on the health and quality of life of people with cystic fibrosis, raising questions about the need for ongoing supportive therapies. This study examines patient-reported outcomes related to ETI and its influence on supportive therapy adherence. An anonymized cross-sectional survey was conducted among 1,589 members of the German Cystic Fibrosis Association to assess treatment burden, and quality of life before and during ETI treatment, focusing on adherence and factors affecting the continuation of supportive therapies. Of the 406 respondents (effective response rate 25.5%), 276 were receiving ETI. Analysis showed increased quality of life and decreased symptoms, with a majority reporting reduced or absent cough (93%) and sputum (91%) following ETI treatment. The desire to reduce therapy burden was high, with 80.4 % reporting a prior or present reduction of supportive therapies, particularly of mucoactive inhalations and antibiotics. Age was a significant factor in therapy adherence, with higher adherence in adolescents than adults. Improved quality of life and better lung function correlated with greater adherence to antibiotic inhalations, while better lung function increased the likelihood of reducing mucoactive therapies. Many people with CF are reducing supportive therapies despite the absence of clear guidelines. Decision-making is influenced by factors such as the desire to lessen therapy burden, reduced symptom severity, and maximizing therapeutic effects. The study highlights the need for comprehensive recommendations and calls for further research to understand the factors involved in therapy adherence and reduction.

Incontinentia pigmenti (IP) is a rare hereditary disorder affecting 1.2 in 100,000 live births, predominantly females. Genetic analysis of IP is complicated by a homologous pseudogene, making conventional short‐read sequencing challenging. While long‐range PCR is typically used to overcome this, skewed X‐inactivation detection can also aid in assigning variants to IKBKG. We employed a comprehensive approach, incorporating whole‐exome sequencing (WES), long‐range PCR, RT‐PCR, X‐inactivation analysis, and nanopore sequencing, to identify and accurately phase a small heterozygous deletion, NM_001099857.5: c.363_367del, p.(Leu122Glyfs ^∗14), in the IKBKG gene in an IP‐affected family. The deletion was initially detected via WES, with skewed X‐inactivation observed in both the proband and her mother. Long‐range PCR specific to IKBKG confirmed the variant’s location in the IKBKG gene, not in the pseudogene. On the RNA level, the variant was undetectable, suggesting nonsense‐mediated decay of the transcript. Nanopore sequencing precisely mapped the variant to IKBKG and analyzed the methylation status of both alleles, confirming the skewed X‐inactivation, with the variant‐carrying allele predominantly inactivated. This demonstrates the nanopore sequencing’s value in genetic diagnosis, enabling precise variant localization and analysis of X chromosome activation status in females with skewed X‐inactivation, aiding in accurate diagnosis and understanding of IP.

Background: Incontinentia pigmenti (IP) is a rare, hereditary multisystemic disorder affecting 1.2 in 100,000 live births, predominantly females. Conventional genetic analyses through short-read sequencing are complicated in case of IP due to the presence of a highly homologous pseudogene. Traditionally, long-range PCR is employed in order to overcome this challenge, however, detection of skewed X-Inactivation can also aid in correctly assign a variant to IKBKG . Methods: We employed a comprehensive multi-method approach, incorporating whole-exome sequencing (WES), long-range PCR, RT-PCR, X-inactivation analysis, and nanopore sequencing of genomic DNA, to identify and accurately phase a small heterozygous deletion NM_001099857.5:c.363_367del, p.(Leu122Glyfs*14) to the IKBKG gene in a family affected with IP. Results: The deletion was initially detected through WES and skewed X-inactivation was observed in both the proband and her mother. Long-range PCR specific to IKBKG was utilized to verify that the variant is located in IKBKG and not in its highly homologous pseudogene. On RNA level, the variant was undetectable, suggesting nonsense-mediated decay (NMD) of the transcript containing the variant. We further utilized nanopore sequencing not only to pinpoint and accurately map the variant to the IKBKG gene but also to analyze methylation status of both alleles. This allowed us to confirm the skewed X-inactivation, with the variant-carrying allele found to be predominantly inactivated. Conclusion: Nanopore sequencing serves as a valuable tool in genetic diagnosis, enabling the precise localization of the variant in either the gene or the pseudogene. Furthermore, in females with skewed X-inactivation, this method facilitates the determination of whether the variant is predominantly located on the activated or inactivated X-chromosome.

Accurate genetic diagnosis is essential for appropriate treatment in cystic fibrosis (CF). Large copy number variants like duplications in the CFTR gene are rare and often classified as variants of uncertain significance (VUSs) due to unknown characteristics of the inserted material, complicating diagnosis and treatment decisions. We identified a previously uncharacterized exon 22 duplication (CFTRdup22) in the CFTR gene in two anamnestically unrelated people with CF, both exhibiting a mild phenotype. Initial classification as a VUS was based on standard genetic testing. We employed a custom next-generation sequencing (NGS) panel to determine the exact breakpoints of the duplication and conducted mRNA sequencing to confirm its effect on splicing. DNA and RNA analyses allowed for precise breakpoint determination, confirming that the duplication was in tandem and the reading frame remained intact. This, as well as a residual CFTRdup22 function of ~30% as measured via intestinal current measurement, is consistent with a clinically milder CF phenotype. Collectively, the precise characterization of the variants’ breakpoints, localization and orientation enabled us to reclassify the variant as likely pathogenic. This study highlights the importance of advanced genetic techniques, such as NGS and breakpoint analysis, in accurately identifying CF-causing variants. It underscores the importance of a comprehensive approach and persistence when suspecting a specific genetic condition. This can aid in reclassifying VUSs, providing a definitive diagnosis for the affected family and enabling appropriate therapeutic interventions, including the use of CFTR modulators.

Article Die GfH-Juniorakademie 2022 – #GfHJAK22 @schlossbuchenau was published on December 1, 2022 in the journal Medizinische Genetik (volume 34, issue 4).