In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells.

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF.

Quantitative Evaluation of CFTR Gene Expression: A Comparison between Relative Quantification by Real-Time PCR and Absolute Quantification by Droplet Digital PCR.

In the precision medicine era of cystic fibrosis (CF), therapeutic interventions, by the so-called modulators, target the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The levels of targetable CFTR proteins are a main variable in the success of patient-specific therapy. In turn, the CFTR protein level depends, at least in part, on the level of CFTR mRNA. Many mechanisms can modulate the CFTR mRNA level, for example, transcriptional rate, stability of the mRNA, epigenetics, and pathogenic variants that can affect mRNA production and degradation. Independently from the causes of variable CFTR mRNA levels, their exact quantitative assessment is of great importance in CF. Methods with high analytical sensitivity, precision, and accuracy are mandatory for the quantitative evaluation aimed at the amelioration of the diagnostic, prognostic, and therapeutic aspects. This paper compares, for the first time, two CFTR gene expression quantification methods: a well-established method for the relative quantification of CFTR mRNA using a real-time PCR and an innovative method for its absolute quantification using a droplet digital PCR. No comprehensive methods for absolute CFTR quantification via droplet digital PCR have been published so far. The accurate quantification of CFTR expression at the mRNA level is a critical step for the personalized therapeutic approaches of CF.

L1077P CFTR pathogenic variant function rescue by Elexacaftor-Tezacaftor-Ivacaftor in cystic fibrosis patient-derived air-liquid interface (ALI) cultures and organoids: in vitro guided personalized therapy of non-F508del patients.

Cystic fibrosis (CF) is caused by defects of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR-modulating drugs may overcome specific defects, such as the case of Trikafta, which is a clinically approved triple combination of Elexacaftor, Tezacaftor and Ivacaftor (ETI) that exhibited a strong ability to rescue the function of the most frequent F508del pathogenic variant even in genotypes with the mutated allele in single copy. Nevertheless, most rare genotypes lacking the F508del allele are still not eligible for targeted therapies. Via the innovative approach of using nasal conditionally reprogrammed cell (CRC) cell-based models that mimic patient disease in vitro, which are obtainable from each patient due to the 100% efficiency of the cell culture establishment, we theratyped orphan CFTR mutation L1077P. Protein studies, Forskolin-induced organoid swelling, and Ussing chamber assays congruently proved the L1077P variant function rescue by ETI. Notably, this rescue takes place even in the context of a single-copy L1077P allele, which appears to enhance its expression. Thus, the possibility of single-allele treatment also arises for rare genotypes, with an allele-specific modulation as part of the mechanism. Of note, besides providing indication of drug efficacy with respect to specific CFTR pathogenic variants or genotypes, this approach allows the evaluation of the response of single-patient cells within their genetic background. In this view, our studies support in vitro guided personalized CF therapies also for rare patients who are nearly excluded from clinical trials.

Theratyping cystic fibrosis in vitro in ALI culture and organoid models generated from patient-derived nasal epithelial conditionally reprogrammed stem cells.

QUESTION: Cystic fibrosis (CF) is due to pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recent improvements have enabled pharmacological therapy aiming at restoring mutated CFTR expression and function. CFTR "modulators" have revolutionised the CF therapeutic landscape, particularly the last approved, Trikafta. This drug combination is indicated by the United States Food and Drug Administration and very recently by the European Medicines Agency for genotypes carrying at least one copy of CFTR with the F508del pathogenic variant. However, several genotypes are not yet eligible for Trikafta treatment. MATERIALS/PATIENTS AND METHODS: We exploited an innovative cellular approach allowing highly efficient in vitro expansion of airway epithelial stem cells (AESCs) through conditional reprogramming from nasal brushing of CF patients. This approach, coupled to the development of AESC-derived personalised disease models, as organoids and air-liquid interface (ALI) cultures, revealed highly suitable for CFTR pharmacological testing. RESULTS AND ANSWER TO THE QUESTION: We fully validated the experimental models and implemented the CFTR functional assays and biochemical CFTR protein characterisation, which allowed the evaluation of the efficacy of clinically available modulators in restoring CFTR maturation and function of each patient-derived "avatar" (theratyping). F508del homozygous genotypes, used as controls, confirmed the higher clinical activity of Trikafta in comparison with older modulators. In addition, Trikafta showed its efficacy on three rare genotypes previously not eligible for treatment with modulators, opening the way to clinical translation. Finally, encouraging results for innovative drug combinations were obtained.

SPEN haploinsufficiency causes a neurodevelopmental disorder overlapping proximal 1p36 deletion syndrome with an episignature of X chromosomes in females.

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.

Refinement of the clinical and mutational spectrum of UBE2A deficiency syndrome.

UBE2A deficiency, that is, intellectual disability (ID) Nascimento type (MIM 300860), is an X-linked syndrome characterized by developmental delay, moderate to severe ID, seizures, dysmorphisms, skin anomalies, and urogenital malformations. Forty affected subjects have been reported thus far, with 31 cases having intragenic UBE2A variants. Here, we report on additional eight affected subjects from seven unrelated families who were found to be hemizygous for previously unreported UBE2A missense variants (p.Glu62Lys, p.Arg95Cys, p.Thr99Ala, and p.Arg135Trp) or small in-frame deletions (p.Val81_Ala83del, and p.Asp101del). A wide phenotypic spectrum was documented in these subjects, ranging from moderate ID associated with mild dysmorphisms to severe features including congenital heart defects (CHD), severe cognitive impairment, and pineal gland tumors. Four variants affected residues (Glu62, Arg95, Thr99 and Asp101) that contribute to stabilizing the structure of the E3 binding domain. The three-residue in-frame deletion, p.Val81_Ala83del, resulted from aberrant processing of the transcript. This variant and p.Arg135Trp mapped to regions of the protein located far from the E3 binding region, and caused variably accelerated protein degradation. By reviewing available clinical information, we revise the clinical and molecular profile of the disorder and document genotype-phenotype correlations. Pineal gland cysts/tumors, CHD and hypogammaglobulinemia emerge as recurrent features.