[3120+1kbdel86kb]+[pN1303K] genotype in an Emirati cystic fibrosis patient: indication of a founder mutation in Palestinian Arabs

Cystic fibrosis (CF) is the most common life-limiting autosomal recessive disorder in Caucasian population. The disease was initially considered to be rare in Middle Eastern countries. 95% of CF in Emirati families is due to two mutations only--p.S549R(T \u3e G) and p.F508del. We report here the case of a patient referred to CF and Respiratory Clinic at Tawam Hospital for cystic fibrosis transmembrane regulator (CFTR) gene screening to ascertain the diagnosis of CF, who was found to carry a unique genotype, signifying the importance of retrieving ancestral histories of patients with monogenic disorders

Combined Computational-Experimental Analyses of CFTR Exon Strength Uncover Predictability of Exon-Skipping Level.

International audienceWith the increased number of identified nucleotide sequence variations in genes, the current challenge is to classify them as disease causing or neutral. These variants of unknown clinical significance can alter multiple processes, from gene transcription to RNA splicing or protein function. Using an approach combining several in silico tools, we identified some exons presenting weaker splicing motifs than other exons in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. These exons exhibit higher rates of basal skipping than exons harboring no identifiable weak splicing signals using minigene assays. We then screened 19 described mutations in three different exons, and identified exon-skipping substitutions. These substitutions induced higher skipping levels in exons having one or more weak splicing motifs. Indeed, this level remained under 2% for exons with strong splicing motifs and could reach 40% for exons having at least one weak motif. Further analysis revealed a functional exon splicing enhancer within exon 3 that was associated with the SR protein SF2/ASF and whose disruption induced exon skipping. Exon skipping was confirmed in vivo in two nasal epithelial cell brushing samples. Our approach, which point out exons with some splicing signals weaknesses, will help spot splicing mutations of clinical relevance

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies

Molecular Diagnosis and Genetic Counseling of Cystic Fibrosis and Related Disorders: New Challenges

Identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and its numerous variants opened the way to fantastic breakthroughs in diagnosis, research and treatment of cystic fibrosis (CF). The current and future challenges of molecular diagnosis of CF and CFTR-related disorders and of genetic counseling are here reviewed. Technological advances have enabled to make a diagnosis of CF with a sensitivity of 99% by using next generation sequencing in a single step. The detection of heretofore unidentified variants and ethnic-specific variants remains challenging, especially for newborn screening (NBS), CF carrier testing and genotype-guided therapy. Among the criteria for assessing the impact of variants, population genetics data are insufficiently taken into account and the penetrance of CF associated with CFTR variants remains poorly known. The huge diversity of diagnostic and genetic counseling indications for CFTR studies makes assessment of variant disease-liability critical. This is especially discussed in the perspective of wide genome analyses for NBS and CF carrier screening in the general population, as future challenges

The Role of Extended CFTR Gene Sequencing in Newborn Screening for Cystic Fibrosis

There has been considerable progress in the implementation of newborn screening (NBS) programs for cystic fibrosis (CF), with DNA analysis being part of an increasing number of strategies. Thanks to advances in genomic sequencing technologies, CFTR-extended genetic analysis (EGA) by sequencing its coding regions has become affordable and has already been included as part of a limited number of core NBS programs, to the benefit of admixed populations. Based on results analysis of existing programs, the values and challenges of EGA are reviewed in the perspective of its implementation on a larger scale. Sensitivity would be increased at best by using EGA as a second tier, but this could be at the expense of positive predictive value, which improves, however, if EGA is applied after testing a variant panel. The increased detection of babies with an inconclusive diagnosis has proved to be a major drawback in programs using EGA. The lack of knowledge on pathogenicity and penetrance associated with numerous variants hinders the introduction of EGA as a second tier, but EGA with filtering for all known CF variants with full penetrance could be a solution. The issue of incomplete knowledge is a real challenge in terms of the implemention of NBS extended to many genetic diseases

Mosaicism in men in hemophilia: is it exceptional? Impact on genetic counselling.

International audienceNo summar

Multiplex allele-specific fluorescent PCR for haplotyping the IVS8 (TG)m(T)n locus in the CFTR gene.: Direct haplotyping of CFTR IVS8 (TG)m(T)n

International audienceBACKGROUND: Precise genotyping of the intron 8 poly(TG) and poly(T) tracts of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is of clinical relevance in CFTR pathology. The (TG)(m) locus influences the penetrance of the (T)(5) allele, which may be associated with male infertility by congenital bilateral absence of the vas deferens (CBAVD) or other CFTR-related disorders (CFTR-RD), in particular in the context of (TG)(12) and (TG)(13). Simple and accurate genotyping of both loci should thus be routinely offered in laboratories. METHODS: We designed a new single test method relying on multiplex allele-specific fluorescent PCR: (T)(5)-, (T)(7)-, and (T)(9)-specific primers, labeled with different fluorophores, in combination with a common primer. Each fluorescent PCR product was identified on a capillary sequencer by its fluorescence color, specific for (T)(n), and size, indicative of the (TG) length. We first validated the assay in 2 different laboratories on 52 DNA samples with already known genotypes. We then evaluated the method prospectively, compared with sequencing, on 62 samples from healthy individuals and 108 samples from patients with CBAVD or other CFTR-RDs. RESULTS: We observed a 100% match in both validation steps. Results found in CBAVD and CFTR-RD patients are in keeping with data in the literature. CONCLUSIONS: The assay proved to be simple, rapid, and accurate for single-test (TG)(m)(T)(n) genotyping and suited for analysis in clinical laboratories

Clinical interpretation of PRSS1 variants in patients with pancreatitis

International audienceSince the description of the PRSS1 gene encoding the cationic trypsinogen as being involved in dominant hereditary pancreatitis, more than 50 PRSS1 variants have been reported. Among the PRSS1 variants that have been classified as pathogenic, some have a high penetrance and others have a low penetrance. Assessing the clinical relevance of PRSS1 variants is often complicated in the absence of functional evidence and interpretation of rare variants is not very easy in clinical practice. The aim of this study was to review the different variants identified in the PRSS1 gene and to classify them according to their degree of deleterious effect. This classification was based on the results of several in vitro experiments and on population data, in comparing the allelic frequency of each variant in patients with pancreatitis and in unaffected individuals. This review should help geneticists and clinicians in charge of patient's care and genetic counseling to interpret molecular results

Con le fondamenta nel Medioevo: Sant'Antonio di Padova a Bologna

L'articolo descrive le scelte relative all'edificazione nei primi anni del XX secolo della chiesa di Sant'Antonio da Padova, uno dei migliori esempi bolognesi di architettura revivalistica. Dopo un primo progetto di Collamarini, l'architetto di fiducia di Rubbiani, il complesso viene compiuto su disegno di Carlo Barberi, restauratore del Duomo di Modena e dell'abbazia di Nonantola, in stile gotico. Le fonti documentarie collocano l'intero percorso edilizio entro i confini del 1904