Short reports

Mild cystic fibrosis and normal or borderline sweat test in patients with the 3849 + 10 kb C → T mutation

Ample data on recessive disorders among Ashkenazi Jews has been gathered and published through the years. The opportunity to integrate molecular records analyzed in actual affected individuals with data derived from population-documented frequencies enables to compare these figures.

We reviewed assumed pathogenic variants reported among patients in the Israeli medical genetic database (IMGD) with a carrier frequency of 1% or more among Ashkenazi Jews in gnomAD. Among the 60 assumed pathogenic variants recorded in IMGD, 15 (25%) had either a disease incidence considerably lower than expected by the calculated carrier frequency (12 variants), or the variant was not characterized in Ashkenazi Jewish patients (three variants).

Possible explanations for the rarity or absence of affected individuals despite high carrier frequency include embryonic lethality, clinical variability, and incomplete and age-related penetrance, in addition to the existence of additional assumed pathogenic variants on the founder haplotype, hypomorphic variants or digenic inheritance.

The discrepancy in actual versus expected number of patients calls for caution upon designing and choosing targeted genes and recessive mutations for carrier screening.

People with cystic fibrosis carrying residual function (RF) mutations are considered to have a mild disease course. This may influence caregivers and patients on how intensive the treatments should be.

Characterize disease severity of patients carrying RF mutations, using the European CF Society Patient Registry (ECFSPR) data.

Demographic, clinical characteristics, lung function and death probability of patients carrying at least one RF mutation were analyzed and compared to patients homozygous to minimal function mutations (MF).

Of the 44,594 eligible patients (median age 19.5 years, IQR 10–29.8), 6,636 (14.6%) carried RF mutations, and 37,958 (85.1%) MF mutations. Patients carrying RF mutations were older, diagnosed at a later age, had lower sweat chloride at diagnosis and better FEV1pp at each age group. However, their FEV1pp declined with age and rates of chronic Pseudomonas aeruginosa increased with age. A significant number of patients with RF had FEV1pp similar to patients with MF at each age group. 4.5% of RF patients were treated with oxygen and 2.61% had a lung transplant. With increasing age, 26.6% of RF patients were treated with pancreatic enzymes associated with a more severe lung disease. RF patients had shortened life spans, with mortality starting around the age of 20 years.

Patients carrying an RF mutations experience a decline of pulmonary function with age, leading to life-shortening. Standard of care therapies and augmenting CFTR function may improve their survival and quality of life.

This paper is the first in a series providing updated guidance on the definition, evaluation and management of people with a Cystic Fibrosis Transmembrane conductance Regulator (CFTR)-Related Disorder (CFTR-RD). The need for this update relates to more precise characterisation of CFTR gene variants and improved assessment of CFTR protein dysfunction. The exercise is co-ordinated by the European CF Society Standards of Care Committee and Diagnostic Network Working Group and involves stakeholder engagement. This first paper was produced by a core group using an extensive literature review and papers graded for their quality. Subsequent wider stakeholder agreement was achieved.

The definition of a CFTR-RD remains “a clinical condition with evidence of CFTR protein dysfunction that does not fulfil the diagnostic criteria for CF”. Clearer guidance on CFTR dysfunction and relevant CFTR variants will be provided. Thresholds for clinical presentations are presented and the paradigm that pathobiological processes may be evident in more than one organ is agreed. In this paper we reflect on the early patient journey, highlighting that CF specialists as well as other relevant specialists should be involved in the care of people with a CFTR-RD.

There is now increased knowledge and experience of newborn screening around the world. There is also a better understanding of CF gene analysis, informed by international databases. This has resulted in a small number of children and adults having their diagnosis of CF reversed. This article illustrates this issue with three cases. It considers how best to tell children and adults with their families, and the reactions that may be encountered. It also discusses practical issues of removing the diagnosis.

Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10 kb C-to-T splicing mutation in the CFTR gene.

We have screened, in FRT cells expressing the 3849+10 kb C-to-T splicing mutation, ~30 2′-O-Methyl-modified phosphorothioate ASOs, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The effect of highly potent ASO candidates on the splicing pattern, protein maturation and CFTR function was further analyzed in well differentiated primary human nasal and bronchial epithelial cells, derived from patients carrying at least one 3849+10 kb C-to-T allele.

A highly potent lead ASO, efficiently delivered by free uptake, was able to significantly increase the level of correctly spliced mRNA and completely restore the CFTR function to wild type levels in cells from a homozygote patient. This ASO led to CFTR function with an average of 43% of wild type levels in cells from various heterozygote patients. Optimized efficiency of the lead ASO was further obtained with 2′-Methoxy Ethyl modification (2′MOE).

The highly efficient splicing modulation and functional correction, achieved by free uptake of the selected lead ASO in various patients, demonstrate the ASO therapeutic potential benefit for CF patients carrying splicing mutations and is aimed to serve as the basis for our current clinical development.

To determine the frequency of cystic fibrosis (CF) carriers among sperm donors in Spain studied through a complete analysis of the CFTR gene and to compare the results with those that would have been obtained by the 4 genotyping panels of the CFTR gene most commonly used as a carrier test in the context of assisted reproduction in our country.

Descriptive observational study.

Private center.

Nine hundred thirty-five sperm donors, from January 2014 to June 2019.

None.

Presence of pathogenic variants in the CFTR gene.

17% of the donors were carriers of at least 1 pathogenic variant in CFTR, with 39 different pathogenic variants detected. Only 4 of these 39 variants (10.27%) would have been detected by the 4 genotyping tests considered, and 22 variants (56.41%) would not have been detected by any of the genotyping tests. The pathogenic variants of the CFTR gene included in the different genotyping tests analyzed vary widely, and <50% are common to all of them.

Although the was not based in the general population, these results show that the use of genotyping tests is associated with a high reproductive risk, because the rate of detection of CF carriers was lower when these panels were applied, in comparison with the complete study of the CFTR gene. We recommend that complete sequencing of the CFTR gene by next-generation sequencing be performed as a screening method for CF in sperm donors.

Portadores de fibrosis quística entre donantes de esperma: análisis completo del gen CFTR versus genotipado CFTR.

Determinar la frecuencia de portadores de fibrosis quística (FQ) entre los donantes de esperma en España estudiados mediante un análisis completo del gen CFTR, y comparar los resultados con los que se habrían obtenido mediante los 4 paneles de genotipado del gen CFTR más utilizados como test de portadores en el contexto de la reproducción asistida en nuestro país.

Estudio observacional descriptivo.

Centro privado.

Novecientos treinta y cinco donantes de esperma, desde enero de 2014 a junio de 2019.

Ninguna.

Presencia de variantes patogénicas en el gen CFTR.

El 17% de los donantes eran portadores de al menos 1 variante patogénica en CFTR, con detección de 39 diferentes variantes patogénicas detectadas. Solamente 4 de estas 39 variantes (10.27%) habrían sido detectadas por las 4 pruebas de genotipado consideradas, y 22 variantes (56.41%) no habrían sido detectadas por ninguna de las pruebas de genotipado. Las variantes patogénicas del gen CFTR incluidas en las diferentes pruebas de genotipado analizadas varían ampliamente, y <50% son comunes a todos ellos.

Aunque no se basaron en la población general, estos resultados muestran que el uso de pruebas de genotipado está asociado con un alto riesgo reproductivo, debido a que la tasa de detección de portadores de FQ fue menor cuando se aplicaron estos paneles, en comparación con el estudio completo del gen CFTR. Recomendamos que la secuenciación completa del gen CFTR mediante secuenciación de última generación (NGS) sea realizada como método de detección de la FQ en donantes de esperma.