Two rare variants that affect the same amino acid in CFTR have distinct responses to ivacaftor

Some residues in the cystic fibrosis transmembrane conductance regulator (CFTR) channel are the site of more than one CFTR variant that cause cystic fibrosis. Here, we investigated the function of S1159F and S1159P, two variants associated with different clinical phenotypes, which affect the same pore-lining residue in transmembrane segment 12 that are both strongly potentiated by ivacaftor when expressed in CFBE41o− bronchial epithelial cells. To study the single-channel behaviour of CFTR, we applied the patch-clamp technique to Chinese hamster ovary cells heterologously expressing CFTR variants incubated at 27°C to enhance channel residence at the plasma membrane. S1159F- and S1159P-CFTR formed Cl− channels activated by cAMP-dependent phosphorylation and gated by ATP that exhibited thermostability at 37°C. Both variants modestly reduced the single-channel conductance of CFTR. By severely attenuating channel gating, S1159F- and S1159P-CFTR reduced the open probability (Po) of wild-type CFTR by ≥75% at ATP (1 mM); S1159F-CFTR caused the greater decrease in Po consistent with its more severe clinical phenotype. Ivacaftor (10–100 nM) doubled the Po of both CFTR variants without restoring Po values to wild-type levels, but concomitantly, ivacaftor decreased current flow through open channels. For S1159F-CFTR, the reduction of current flow was marked at high (supersaturated) ivacaftor concentrations (0.5–1 μM) and voltage-independent, identifying an additional detrimental action of elevated ivacaftor concentrations. In conclusion, S1159F and S1159P are gating variants, which also affect CFTR processing and conduction, but not stability, necessitating the use of combinations of CFTR modulators to optimally restore their channel activity

Things come in threes: A new complex allele and a novel deletion within the CFTR gene complicate an accurate diagnosis of cystic fibrosis

Background: Despite consolidated guidelines, the clinical diagnosis and prognosis of cystic fibrosis (CF) is still challenging mainly because of the extensive phenotypic heterogeneity and the high number of CFTR variants, including their combinations as complex alleles. Results: We report a family with a complicated syndromic phenotype, which led to the suspicion not only of CF, but of a dominantly inherited skeletal dysplasia (SD). Whereas the molecular basis of the SD was not clarified, segregation analysis was central to make a correct molecular diagnosis of CF, as it allowed to identify three CFTR variants encompassing two known maternal mutations and a novel paternal microdeletion. Conclusion: This case well illustrates possible pitfalls in the clinical and molecular diagnosis of CF; presence of complex phenotypes deflecting clinicians from appropriate CF recognition, and/or identification of two mutations assumed to be in trans but with an unconfirmed status, which underline the importance of an in-depth molecular CFTR analysis

Sources of Variation in Sweat Chloride Measurements in Cystic Fibrosis

Rationale: Expanding the use of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators and correctors for the treatment of cystic fibrosis (CF) requires precise and accurate biomarkers. Sweat chloride concentration provides an in vivo assessment of CFTR function, but it is unknown the degree to which CFTR mutations account for sweat chloride variation

Achados clínicos em crianças e adolescentes com fibrose cística e sua relação com variáveis socioeconômicas e moleculares

Introdução: A Fibrose cística (FC) é uma doença genética autossômica recessiva limitadora de vida. Mais de 2000 mutações no gene CFTR já foram identificadas. A abrangência de manifestações clínicas na FC varia, tanto em relação ao número de órgãos afetados, quanto à gravidade da doença, sem uma correlação genótipo-fenótipo forte o suficiente para explicar tal variabilidade. É possível que outros fatores genéticos ou mesmo fatores não genéticos influenciem os fenótipos de forma significativa. Objetivo: Avaliar a correlação dos achados moleculares no gene CFTR e variáveis socioeconômicas com achados clínicos de crianças e adolescentes com fibrose cística acompanhados no Hospital de Clínicas de Porto Alegre (HCPA). Metodologia: Os pacientes de até 18 anos foram recrutados prospectivamente a partir do ambulatório de Pneumologia Pediátrica específico para Fibrose Cística do HCPA. Durante a avaliação dos pacientes, além da avaliação clínica e laboratorial realizadas na rotina do serviço foi aplicado questionário sobre dados socioeconômicos da Associação Brasileira de Empresas de Pesquisa (ABEP) conforme o Critério Brasil 2018. Os pacientes, que no momento do recrutamento ainda não tinham realizado a dosagem de elastase fecal como parte de sua avaliação de rotina, foram convidados a fazer o exame como parte do estudo. Pacientes que não tinham seu genótipo estabelecido foi realizado sequenciamento de nova geração (NGS). Resultados: No total foram alocados 87 pacientes com mediana de idade de 8 (4-14) anos. Os pacientes foram divididos em cinco grupos conforme seu genótipo. A mutação mais frequente encontrada foi a p.Phe508del presente em 57.9% dos alelos estudados. Além da p.Phe508del outras seis mutações tiveram frequência acima de 2%. A maioria dos pacientes foram insuficientes pancreáticos (95,4%) com mediana da elastase fecal de 15 (15-15) mcg/g. Os pacientes encontravam-se nutricionalmente com escore Z para Índice de Massa Corporal (Z- IMC) de 0.14±0.98 e para altura de -1.00 (-1.65 - -0.35). O nível médio de albumina foi de 4.42±0.347g/dL. A média do escore Z para valores de função pulmonar do VEF1 dos 52 pacientes com espirometrias que cumpriam critérios de aceitabilidade foi de -1.05 (-2.29 - -0.02) e do CVF -0.927±2.67. Houve correlação significativa entre albumina e Z-IMC (0,32; p < 0.01). Ambas as variáveis apresentaram correlação com a idade, de forma que quanto mais velho o paciente menor o Z-IMC (-0.35; p<0.01) e a albumina (-0.23; p <0.05). Quando comparados os dados do Escore ultrassonográfico Hepático de Williams houve diferença significativa entre os grupos (p= 0.0). Houve diferença limítrofe entre os grupos nos dados de radiografia de tórax (p = 0.051). Pacientes com níveis de albumina maior foram associados a maiores valores de Z-VEF1 (0.4; p<0.01). Pacientes em que a pontuação da ABEP foi maior tiveram um Z-Altura maior também (0.26; p<0.05). Conclusão: Poucos desfechos avaliados no estudo relacionaram-se com o genótipo. Outras variáveis genéticas podem estar envolvidas. Pacientes menos favorecidos socioeconomicamente apresentaram menor estatura demonstrando uma relação ambiental com a apresentação clínica da doença.Introduction: Cystic fibrosis (CF) is a life-limiting autosomal recessive genetic disease. More than 2000 mutations in the CFTR gene have been identified. The scope of clinical manifestations in CF varies, both in relation to the number of affected organs and the severity of the disease not existing a strong genotype-phenotype correlation to explain such variability. It is possible that other genetic factors or even non-genetic factors influence the phenotypes significantly. Objective: To evaluate the correlation of molecular findings in the CFTR gene and socioeconomic variables with clinical findings of children and adolescents with cystic fibrosis followed up at Hospital de Clínicas de Porto Alegre (HCPA). Methodology: Patients aged up to 18 years old were prospectively recruited from the HCPA Pediatric Pulmonology outpatient clinic. During the patients visit to the clinic, in addition to the clinical and laboratory evaluation carried out in the routine, a questionnaire was applied on socioeconomic data from the Brazilian Association of Research Companies (ABEP) according to the 2018 Brazil criteria. Patients who, at the time of recruitment, had not yet undergone fecal elastase measurement as part of their routine assessment, they were invited to have it test it as part of the study. Patients whose genotype was not establish were sequence through new generations sequencing (NGS). Results: Eighty-seven patients were included in the study. Patients were divided into five groups according to their genotype. The most frequent mutation found was p.Phe508del present in approximately 57.9% of the alleles studied. In addition to p.Phe508del, six other mutations had a frequency above 2%. Most patients were pancreatic insufficient (95.4%) with a median fecal elastase of 15 (15-15) mcg/g. Patients had Body Mass Index Z score (Z-BMI) of 0.14 ± 0.98 and height Z score of -1.00 (-1.65 - -0.35). The average level of albumin was 4.42 ± 0.347g / dL. The mean Z score for pulmonary function values of FEV1 was -1.05 (-2.29 - -0.02) and for CVF -0.92 ± 2.67. There was a significant correlation between albumin and Z-BMI (0.32; p <0.01). Both variables correlated with age, so that the older the patient, the lower the Z-BMI (-0.35; p <0.01) and albumin (-0.23; p <0.05). When comparing the Williams hepatic ultrasound Score data, there was a significant difference between the groups (p=0.03). There was a borderline difference between the groups in the chest X-ray Schwachman score (p=0.05). Patients with higher albumin levels were associated with higher Z-FEV1 values (0.4; p <0.01). Patients whose ABEP score was higher also had a higher Z-Height (0.26; p <0.05). Conclusion: Few outcomes assessed in the study were related to the genotype. Other genetic variables may be involved. Less socio-economically favored patients had shorter stature demonstrating an environmental relationship with the clinical presentation of the disease

Genome-wide association meta-analysis identifies five modifier loci of lung disease severity in cystic fibrosis

The identification of small molecules that target specific CFTR variants has ushered in a new era of treatment for cystic fibrosis (CF), yet optimal, individualized treatment of CF will require identification and targeting of disease modifiers. Here we use genome-wide association analysis to identify genetic modifiers of CF lung disease, the primary cause of mortality. Meta-analysis of 6,365 CF patients identifies five loci that display significant association with variation in lung disease. Regions on chr3q29 (MUC4/MUC20; P=3.3 × 10−11), chr5p15.3 (SLC9A3; P=6.8 × 10−12), chr6p21.3 (HLA Class II; P=1.2 × 10−8) and chrXq22-q23 (AGTR2/SLC6A14; P=1.8 × 10−9) contain genes of high biological relevance to CF pathophysiology. The fifth locus, on chr11p12-p13 (EHF/APIP; P=1.9 × 10−10), was previously shown to be associated with lung disease. These results provide new insights into potential targets for modulating lung disease severity in CF

Standards of care for CFTR variant-specific therapy (including modulators) for people with cystic fibrosis

Cystic fibrosis (CF) has entered the era of variant-specific therapy, tailored to the genetic variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators, the first variant-specific therapy available, have transformed the management of CF.The latest standards of care from the European CF Society (2018) did not include guidance on variant-specific therapy, as CFTR modulators were becoming established as a novel therapy. We have produced interim standards to guide healthcare professionals in the provision of variant-specific therapy for people with CF.Here we provide evidence-based guidance covering the spectrum of care, established using evidence from systematic reviews and expert opinion. Statements were reviewed by key stakeholders using Delphi methodology, with agreement (≥80%) achieved for all statements after one round of consultation. Issues around accessibility are discussed and there is clear consensus that all eligible people with CF should have access to variant-specific therapy

Exploring parental cystic fibrosis disclosure to well children.

Cystic fibrosis (CF), a genetic disease and chronic illness, affects multiple organ systems and requires exceptional medical care and treatment. Few studies have assessed the diagnosis disclosure process to well children when their sibling(s) have CF, and none have evaluated the association between parental knowledge of CF and the disclosure of CF. The objectives of this study were to assess parental understanding of CF, demonstrate the most commonly shared topics and their frequencies of discussion with well children, and identify associations between parental understanding of CF and aspects of the disclosure process to well children. Parents were recruited from CF support organizations and asked to complete an online, anonymous survey. Individuals were eligible to participate in the study if they had at least one living child with CF and at least one living child without CF. Completed surveys from 48 individuals revealed that most parents began discussing a sibling\u27s diagnosis of CF with the first-born well child at 5.4 years old. Topics related to CF were discussed openly and as needed with their well children (n = 44). The most frequently discussed topic, and the topic ranked most important (1.93 of 5, SD: 1.17) by 40 participants (90.9%), was medical concerns and treatment for CF. Fewer parents (n = 18, 40.9%) reported discussing the financial impact of CF, and many ranked this as least important to share (4.64 of 5, SD: 0.75). The CF knowledge assessment revealed that participants were well-informed about CF, with a mean total score of 8.9/10 (SD: 0.91). There were no associations between CF knowledge assessment scores, education level, income, and the topics discussed with well children. These results can be utilized by genetic counselors and other healthcare specialists in discussion with parents about the disclosure process of a diagnosis of CF to well children