Search

BACKGROUND: The t(13;22) Robertsonian translocation constitutes a rare form of rearrangement between acrocentric human chromosomes. Most of the meiotic segregation studies of human Robertsonian translocations have been performed on common t(13;14) and t(14;21) translocations. Analysis of the chromosomal constitution in sperm of Robertsonian translocation carriers is of great interest for assessing the risk of unbalanced forms and adapting genetic counselling. In the present study, we present the first meiotic segregation study of a t(13;22) Robertsonian translocation in human sperm. METHODS: A total of 11 787 sperm nuclei were scored using two distinct FISH labelling techniques, i.e. the locus-specific probes (LSI) method and the whole chromosome painting (WCP) technique. RESULTS: The frequency of normal or balanced sperm resulting from alternate meiotic segregation was 86%. Incidences of unbalanced complements resulting from adjacent segregation modes were 12.79% and 14.36% in LSI and WCP assays, respectively. No significant excess of nullisomy or disomy for the affected chromosomes was observed. CONCLUSIONS: Similar results in segregation were obtained with the two techniques, demonstrating the efficiency of the two strategies for the direct segregation analysis of Roberstsonian translocations. The results obtained indicated a moderate meiotic production of imbalance. This study shows that the rare Robertsonian translocation (13;22) displays a similar distribution of balanced and unbalanced sperm patterns as the common Robertsonian translocations previously studied. This suggests that the behaviour of acrocentric chromosomes was similar in all cases of centric fusion. [ABSTRACT FROM AUTHOR]

Background: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy., Methods: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs., Results: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested., Conclusion: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments., Competing Interests: Conflict of Interest Statement The authors declare no conflicts of interest., (Copyright © 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: Newborn screening for Cystic Fibrosis (CF) is associated with situations where the diagnosis of CF or CFTR related disorders (CFTR-RD) cannot be clearly ruled out., Materials/patients and Methods: We report a case series of 23 children with unconclusive diagnosis after newborn screening for CF and a mean follow-up of 7.7 years (4-13). Comprehensive investigations including whole CFTR gene sequencing, in vivo intestinal current measurement (ICM), nasal potential difference (NPD), and in vitro functional studies of variants of unknown significance, helped to reclassify the patients., Results: Extensive genetic testing identified, in trans with a CF causing mutation, variants with varying clinical consequences and 3 variants of unknown significance (VUS). Eighteen deep intronic variants were identified by deep resequencing of the whole CFTR gene in 13 patients and were finally considered as non-pathogenic. All patients had normal CFTR dependent chloride transport in ICM. NPD differentiated 3 different profiles: CF-like tracings qualifying the patients as CF, such as F508del/D1152H patients; normal responses, suggesting an extremely low likelihood of developing a CFTR-RD such as F508del/TG11T5 patients; partial CFTR dysfunction above 20% of the normal, highlighting a remaining risk of developing CFTR-RD such as F508del/F1052V patients. The 3 VUS were reclassified as variant with defective maturation (D537N), defective expression (T582I) or with no clinical consequence (M952T)., Conclusion: This study demonstrates the usefulness of combining genetic and functional investigations to assess the possibility of evolving to CF or CFTR-RD in babies with inconclusive diagnosis at neonatal screening., Competing Interests: Declaration of Competing Interest All authors have disclosed their conflict of Interest., (Copyright © 2021. Published by Elsevier B.V.)

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing loss combined with retinitis pigmentosa, and in some cases, vestibular areflexia. Three clinical subtypes are distinguished, and MYO7A and USH2A represent the two major causal genes involved in Usher type I, the most severe form, and type II, the most frequent form, respectively. Massively parallel sequencing was performed on a cohort of patients in the context of a molecular diagnosis to confirm clinical suspicion of Usher syndrome. We report here 231 pathogenic MYO7A and USH2A genotypes identified in 73 Usher type I and 158 Usher type II patients. Furthermore, we present the ACMG classification of the variants, which comprise all types. Among them, 68 have not been previously reported in the literature, including 12 missense and 16 splice variants. We also report a new deep intronic variant in USH2A . Despite the important number of molecular studies published on these two genes, we show that during the course of routine genetic diagnosis, undescribed variants continue to be identified at a high rate. This is particularly pertinent in the current era, where therapeutic strategies based on DNA or RNA technologies are being developed.

Cystic fibrosis (CF) is a chronic genetic disease that mainly affects the respiratory and gastrointestinal systems. No curative treatments are available, but the follow-up in specialized centers has greatly improved the patient life expectancy. Robust biomarkers are required to monitor the disease, guide treatments, stratify patients, and provide outcome measures in clinical trials. In the present study, we outline a strategy to select putative DNA methylation biomarkers of lung disease severity in cystic fibrosis patients. In the discovery step, we selected seven potential biomarkers using a genome-wide DNA methylation dataset that we generated in nasal epithelial samples from the MethylCF cohort. In the replication step, we assessed the same biomarkers using sputum cell samples from the MethylBiomark cohort. Of interest, DNA methylation at the cg11702988 site ( ATP11A gene) positively correlated with lung function and BMI, and negatively correlated with lung disease severity, P. aeruginosa chronic infection, and the number of exacerbations. These results were replicated in prospective sputum samples collected at four time points within an 18-month period and longitudinally. To conclude, (i) we identified a DNA methylation biomarker that correlates with CF severity, (ii) we provided a method to easily assess this biomarker, and (iii) we carried out the first longitudinal analysis of DNA methylation in CF patients. This new epigenetic biomarker could be used to stratify CF patients in clinical trials.

Cystic fibrosis (CF), a genetic disorder, is characterized by chronic lung disease. Small non-coding RNAs are key regulators of gene expression and participate in various processes, which are dysregulated in CF; however, they remain poorly studied. Here, we determined the complete microRNAs (miRNAs) expression pattern in three CF ex vivo models. The miRNA profiles of air-liquid interface cultures of airway epithelia (bronchi, nasal cells, and nasal polyps) samples from patients with CF and non-CF controls were obtained by deep sequencing. Compared with non-CF controls, several miRNAs were deregulated in CF samples; for instance, miR-181a-5p and the miR-449 family were upregulated. Moreover, mature miRNAs often showed variations (i.e. isomiRs) relative to their reference sequence, such as miR-101, suggesting that miRNAs consist of heterogeneous repertoires of multiple isoforms with different effects on gene expression. Analysis of miR-181a-5p and miR-101-3p roles indicated that they regulate the expression of WISP1, a key component of cell proliferation/migration programs. We showed that miR-101 and miR-181a-5p participated in aberrant recapitulation of wound healing programs by controlling WISP1 mRNA and protein level. Our miRNA expression data bring new insights into CF physiopathology and define new potential therapeutic targets in CF. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The Duchenne muscular dystrophy (DMD) gene has a complex expression pattern regulated by multiple tissue-specific promoters and by alternative splicing (AS) of the resulting transcripts. Here, we used an RNAi-based approach coupled with DMD-targeted RNA-seq to identify RNA-binding proteins (RBPs) that regulate splicing of its skeletal muscle isoform (Dp427m) in a human muscular cell line. A total of 16 RBPs comprising the major regulators of muscle-specific splicing events were tested. We show that distinct combinations of RBPs maintain the correct inclusion in the Dp427m of exons that undergo spatio-temporal AS in other dystrophin isoforms. In particular, our findings revealed the complex networks of RBPs contributing to the splicing of the two short DMD exons 71 and 78, the inclusion of exon 78 in the adult Dp427m isoform being crucial for muscle function. Among the RBPs tested, QKI and DDX5/DDX17 proteins are important determinants of DMD exon inclusion. This is the first large-scale study to determine which RBP proteins act on the physiological splicing of the DMD gene. Our data shed light on molecular mechanisms contributing to the expression of the different dystrophin isoforms, which could be influenced by a change in the function or expression level of the identified RBPs.

Background: Duchenne muscular dystrophy (DMD) is a rare and severe X-linked muscular dystrophy in which the standard of care with variable outcome, also due to different drug response, is chronic off-label treatment with corticosteroids (CS). In order to search for SNP biomarkers for corticosteroid responsiveness, we genotyped variants across 205 DMD-related genes in patients with differential response to steroid treatment., Methods and Findings: We enrolled a total of 228 DMD patients with identified dystrophin mutations, 78 of these patients have been under corticosteroid treatment for at least 5 years. DMD patients were defined as high responders (HR) if they had maintained the ability to walk after 15 years of age and low responders (LR) for those who had lost ambulation before the age of 10 despite corticosteroid therapy. Based on interactome mapping, we prioritized 205 genes and sequenced them in 21 DMD patients (discovery cohort or DiC = 21). We identified 43 SNPs that discriminate between HR and LR. Discriminant Analysis of Principal Components (DAPC) prioritized 2 response-associated SNPs in the TNFRSF10A gene. Validation of this genotype was done in two additional larger cohorts composed of 46 DMD patients on corticosteroid therapy (validation cohorts or VaC1), and 150 non ambulant DMD patients and never treated with corticosteroids (VaC2). SNP analysis in all validation cohorts ( N = 207) showed that the CT haplotype is significantly associated with HR DMDs confirming the discovery results., Conclusion: We have shown that TNFRSF10A CT haplotype correlates with corticosteroid response in DMD patients and propose it as an exploratory CS response biomarker., (Copyright © 2020 Passarelli, Selvatici, Carrieri, Di Raimo, Falzarano, Fortunato, Rossi, Straub, Bushby, Reza, Zharaieva, D’Amico, Bertini, Merlini, Sabatelli, Borgiani, Novelli, Messina, Pane, Mercuri, Claustres, Tuffery-Giraud, Aartsma-Rus, Spitali, T’Hoen, Lochmüller, Strandberg, Al-Khalili, Kotelnikova, Lebowitz, Schwartz, Muntoni, Scapoli and Ferlini.)

Duchenne muscular dystrophy (DMD) is caused by pathogenic variants in the DMD gene leading to the lack of dystrophin. Variability in the disease course suggests that other factors influence disease progression. With this study we aimed to identify genetic factors that may account for some of the variability in the clinical presentation. We compared whole-exome sequencing (WES) data in 27 DMD patients with extreme phenotypes to identify candidate variants that could affect disease progression. Validation of the candidate SNPs was performed in two independent cohorts including 301 (BIO-NMD cohort) and 109 (CINRG cohort of European ancestry) DMD patients, respectively. Variants in the Tctex1 domain containing 1 (TCTEX1D1) gene on chromosome 1 were associated with age of ambulation loss. The minor alleles of two independent variants, known to affect TCTEX1D1 coding sequence and induce skipping of its exon 4, were associated with earlier loss of ambulation. Our data show that disease progression of DMD is affected by a new locus on chromosome 1 and demonstrate the possibility to identify genetic modifiers in rare diseases by studying WES data in patients with extreme phenotypes followed by multiple layers of validation.

Cystic fibrosis (CF) is a rare genetic disease that affects the respiratory and digestive systems. Lung disease is variable among CF patients and associated with the development of comorbidities and chronic infections. The rate of lung function deterioration depends not only on the type of mutations in CFTR, the disease-causing gene, but also on modifier genes. In the present study, we aimed to identify genes and pathways that (i) contribute to the pathogenesis of cystic fibrosis and (ii) modulate the associated comorbidities. We profiled blood samples in CF patients and healthy controls and analyzed RNA-seq data with Weighted Gene Correlation Network Analysis (WGCNA). Interestingly, lung function, body mass index, the presence of diabetes, and chronic P. aeruginosa infections correlated with four modules of co-expressed genes. Detailed inspection of networks and hub genes pointed to cell adhesion, leukocyte trafficking and production of reactive oxygen species as central mechanisms in lung function decline and cystic fibrosis-related diabetes. Of note, we showed that blood is an informative surrogate tissue to study the contribution of inflammation to lung disease and diabetes in CF patients. Finally, we provided evidence that WGCNA is useful to analyze-omic datasets in rare genetic diseases as patient cohorts are inevitably small., Competing Interests: The authors have declared that no competing interests exist.

Exome sequencing used for molecular diagnosis of Mendelian disorders considerably increases the number of missense variants of unclear significance, whose pathogenicity can be assessed by a variety of prediction tools. As the performance of algorithms may vary according to the datasets, complementary specific resources are needed to improve variant interpretation. As a model, we were interested in the cystic fibrosis transmembrane conductance regulator gene (CFTR) causing cystic fibrosis, in which at least 40% of missense variants are reported. Cystic fibrosis missense analysis (CYSMA) is a new web server designed for online estimation of the pathological relevance of CFTR missense variants. CYSMA generates a set of computationally derived data, ranging from evolutionary conservation to functional observations from three-dimensional structures, provides all available allelic frequencies, clinical observations, and references for functional studies. Compared to software classically used in analysis pipelines on a dataset of 141 well-characterized missense variants, CYSMA was the most efficient tool to discriminate benign missense variants, with a specificity of 85%, and very good sensitivity of 89%. These results suggest that such integrative tools could be adapted to numbers of genes involved in Mendelian disorders to improve the interpretation of missense variants identified in the context of diagnosis., (© 2019 Wiley Periodicals, Inc.)

ATP8A2-related disorders are autosomal recessive conditions that associate encephalopathy with or without hypotonia, psychomotor delay, abnormal movements, chorea, tremor, optic atrophy and cerebellar atrophy (CARMQ4). Through a multi-centric collaboration, we identified six point mutations (one splice site and five missense mutations) involving ATP8A2 in six individuals from five families. Two patients from one family with the homozygous p.Gly585Val mutation had a milder presentation without encephalopathy. Expression and functional studies of the missense mutations demonstrated that protein levels of four of the five missense variants were very low and lacked phosphatidylserine-activated ATPase activity. One variant p.Ile215Leu, however, expressed at normal levels and displayed phospholipid-activated ATPase activity similar to the non-mutated protein. We therefore expand for the first time the phenotype related to ATP8A2 mutations to less severe forms characterized by cerebellar ataxia without encephalopathy and suggest that ATP8A2 should be analyzed for all cases of syndromic or non-syndromic recessive or sporadic ataxia.

Background: The CFTR genotype remains incomplete in 1% of Cystic Fibrosis (CF) cases, because only one or no disease-causing variants is detected after extended analysis. This fraction is probably higher in CFTR-Related Disorders (CFTR-RD). Deep-intronic CFTR variants are putative candidates to fill this gap. However, the recurrence, phenotypic spectrum and full molecular characterization of newly reported variants are unknown., Methods: Minigenes and analysis of CFTR transcripts in nasal epithelial cells were used to determine the impact on CFTR splicing of intronic variants that we previously identified by next generation sequencing of the whole CFTR locus. Phenotypic data were collected in 19 patients with CF and CFTR-RD, in whom one of the deep intronic variants has been detected., Results: Three deep-intronic variants promoted the inclusion of pseudo-exons (PE) in the CFTR transcript, hindering the synthesis of a functional protein. The c.2989-313A > T variant, detected in four patients with CF or CFTR-RD from three different families, led to the inclusion of a 118 bp PE. The c.3469-1304C > G variant promoted the inclusion of a 214 bp-PE and was identified in five patients with CF from four families. Haplotype analysis confirmed that this variant was associated with one CF chromosome of African origin. The most represented variant in our cohort was the c.3874-4522A > G, detected in 10 patients with various phenotypes, from male infertility to CF with pancreatic insufficiency., Conclusion: These three deep intronic CFTR variants are associated with a large phenotypic spectrum, including typical CF. They should be included in CF diagnostic testing and carrier screening strategies., (Copyright © 2018. Published by Elsevier B.V.)

Background: Analysis of cell-free fetal DNA in maternal plasma is very promising for early diagnosis of monogenic diseases. However, it has been limited by the need to set up patient- or disease-specific custom-made approaches. Here we propose a universal test based on fluorescent multiplex PCR and size fragment analysis for an indirect diagnosis of cystic fibrosis (CF)., Methods: The test, based on haplotyping, includes nine intra- and extragenic short tandem repeats of the CFTR locus, the coamplification of p.Phe508del (the most frequent mutation in CF patients worldwide), and a specific SRY sequence. The assay is able to determine the inherited paternal allele., Results: Our simple approach was successfully applied to 30 couples and provided clear results from the maternal plasma. The mean rate of informative markers was sufficient to propose it for use in indirect diagnosis., Conclusions: This noninvasive prenatal diagnosis test, focused on indirect diagnosis of CF, offers many advantages over current methods: it is simple, rapid, and cost-effective. It allows for the testing of a large number of couples with high risk of CF, whatever the familial mutation of the CFTR gene. It provides an alternative method to reduce the number of invasive tests., (© 2018 S. Karger AG, Basel.)

Aim: To assess whether DNA methylation levels account for the noninherited phenotypic variations observed among cystic fibrosis (CF) patients., Patients & Methods: Using the 450 K BeadChip, we profiled DNA methylation in nasal epithelial cells collected from 32 CF patients and 16 controls., Results: We detected substantial DNA methylation differences up to 55% (median β change 0.13; IQR: 0.15-0.11) between CF patients and controls. DNA methylation levels differed between mild and severe CF patients and correlated with lung function at 50 CpG sites., Conclusion: In CF samples, dynamic changes of DNA methylation occurred in genes responsible for the integrity of the epithelium and the inflammatory and immune responses, were prominent in transcriptionally active genomic regions and were over-represented in enhancers active in lung tissues. ( Clinicaltrials.gov NCT02884622).

Myopathies and muscular dystrophies (M-MDs) are genetically heterogeneous diseases, with >100 identified genes, including the giant and complex titin (TTN) and nebulin (NEB) genes. Next-generation sequencing technology revolutionized M-MD diagnosis and revealed high frequency of TTN and NEB variants. We developed a next-generation sequencing diagnostic strategy targeted to the coding sequences of 135 M-MD genes. Comparison of two targeted capture technologies (SeqCap EZ Choice library capture kit and Nextera Rapid Capture Custom Enrichment kit) and of two whole-exome sequencing kits (SureSelect V5 and TruSeq RapidExome capture) revealed best coverage with the SeqCap EZ Choice protocol. A marked decrease in coverage was observed with the other kits, affecting mostly the first exons of genes and the repeated regions of TTN and NEB. Bioinformatics analysis strategy was fine-tuned to achieve optimal detection of variants, including small insertions/deletions (INDELs) and copy number variants (CNVs). Analysis of a cohort of 128 patients allowed the detection of 52 substitutions, 13 INDELs (including a trinucleotide repeat expansion), and 3 CNVs. Two INDELs were localized in the repeated regions of NEB, suggesting that these mutations may be frequent but underestimated. A large deletion was also identified in TTN that is, to our knowledge, the first published CNV in this gene., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Many European laboratories offer molecular genetic analysis of the CFTR gene using a wide range of methods to identify mutations causative of cystic fibrosis (CF) and CFTR-related disorders (CFTR-RDs). Next-generation sequencing (NGS) strategies are widely used in diagnostic practice, and CE marking is now required for most in vitro diagnostic (IVD) tests in Europe. The aim of this multicenter study, which involved three European laboratories specialized in CF molecular analysis, was to evaluate the performance of Multiplicom's CFTR MASTR Dx kit to obtain CE-IVD certification., Methods: A total of 164 samples, previously analyzed with well-established "reference" methods for the molecular diagnosis of the CFTR gene, were selected and re-sequenced using the Illumina MiSeq benchtop NGS platform. Sequencing data were analyzed using two different bioinformatic pipelines. Annotated variants were then compared to the previously obtained reference data., Results and Conclusions: The analytical sensitivity, specificity and accuracy rates of the Multiplicom CFTR MASTR assay exceeded 99%. Because different types of CFTR mutations can be detected in a single workflow, the CFTR MASTR assay simplifies the overall process and is consequently well suited for routine diagnostics.

RORα, the RAR-related orphan nuclear receptor alpha, is essential for cerebellar development. The spontaneous mutant mouse staggerer, with an ataxic gait caused by neurodegeneration of cerebellar Purkinje cells, was discovered two decades ago to result from homozygous intragenic Rora deletions. However, RORA mutations were hitherto undocumented in humans. Through a multi-centric collaboration, we identified three copy-number variant deletions (two de novo and one dominantly inherited in three generations), one de novo disrupting duplication, and nine de novo point mutations (three truncating, one canonical splice site, and five missense mutations) involving RORA in 16 individuals from 13 families with variable neurodevelopmental delay and intellectual disability (ID)-associated autistic features, cerebellar ataxia, and epilepsy. Consistent with the human and mouse data, disruption of the D. rerio ortholog, roraa, causes significant reduction in the size of the developing cerebellum. Systematic in vivo complementation studies showed that, whereas wild-type human RORA mRNA could complement the cerebellar pathology, missense variants had two distinct pathogenic mechanisms of either haploinsufficiency or a dominant toxic effect according to their localization in the ligand-binding or DNA-binding domains, respectively. This dichotomous direction of effect is likely relevant to the phenotype in humans: individuals with loss-of-function variants leading to haploinsufficiency show ID with autistic features, while individuals with de novo dominant toxic variants present with ID, ataxia, and cerebellar atrophy. Our combined genetic and functional data highlight the complex mutational landscape at the human RORA locus and suggest that dual mutational effects likely determine phenotypic outcome., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Introduction: Cystic Fibrosis is among the first diseases to have general population genetic screening tests and one of the most common indications of prenatal and preimplantation genetic diagnosis for single gene disorders. During the past twenty years, thanks to the evolution of diagnostic techniques, our knowledge of CFTR genetics and pathophysiological mechanisms involved in cystic fibrosis has significantly improved. Areas covered: Sanger sequencing and quantitative methods greatly contributed to the identification of more than 2,000 sequence variations reported worldwide in the CFTR gene. We are now entering a new technological age with the generalization of high throughput approaches such as Next Generation Sequencing and Droplet Digital PCR technologies in diagnostics laboratories. These powerful technologies open up new perspectives for scanning the entire CFTR locus, exploring modifier factors that possibly influence the clinical evolution of patients, and for preimplantation and prenatal diagnosis. Expert commentary: Such breakthroughs would, however, require powerful bioinformatics tools and relevant functional tests of variants for analysis and interpretation of the resulting data. Ultimately, an optimal use of all those resources may improve patient care and therapeutic decision-making.

This study provides an overview of preimplantation genetic diagnosis (PGD) for single gene diseases and the management of expanding indications in the context of a fully financially covered service at Montpellier's regional hospital centre. Within the framework of a restrictive law ruling PGD in France, only the parental genetic risk can be studied in embryos (concurrent aneuploidy screening is not allowed). PCR-based techniques were developed combining mutation detection and closely linked short tandem repeat markers within or flanking the affected genes, and set up more than 100 different robust fluorescent multiplex assays for 61 monogenic disorders. This strategy was used to analyse blastomeres from cleavage-stage embryos. Overall, 893 cycles were initiated in 384 couples; 727 cycles proceeded to oocyte retrieval and 608 cycles to embryo transfer, resulting in 184 deliveries. Clinical pregnancy rate per transfer, implantation and miscarriage rates were 33.6%, 25.1% and 8.8%, respectively. Our PGD programme resulted in the birth of 214 healthy babies for 162 out of 358 couples (45.3%), constituting a relevant achievement within an organizational framework that does not allow aneuploidy screening but provides equal access to PGD, both geographically and socioeconomically. This is a rare example of a fully free-of-charge PGD service., (Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Mutation-induced exon skipping in the DMD gene can modulate the severity of the phenotype in patients with Duchenne or Becker Muscular Dystrophy. These alternative splicing events are most likely the result of changes in recruitment of splicing factors at cis-acting elements in the mutated DMD pre-mRNA. The identification of proteins involved can be achieved by an affinity purification procedure. Here, we provide a detailed protocol for the in vitro RNA binding assay that we routinely apply to explore molecular mechanisms underlying splicing defects in the DMD gene. In vitro transcribed RNA probes containing either the wild type or mutated sequence are oxidized and bound to adipic acid dihydrazide-agarose beads. Incubation with a nuclear extract allows the binding of nuclear proteins to the RNA probes. The unbound proteins are washed off and then the specifically RNA-bound proteins are released from the beads by an RNase treatment. After separation by SDS-PAGE, proteins that display differential binding affinities for the wild type and mutant RNA probes are identified by mass spectrometry.

Hearing loss is the most common sensory disorder and because of its high genetic heterogeneity, implementation of Massively Parallel Sequencing (MPS) in diagnostic laboratories is greatly improving the possibilities of offering optimal care to patients. We present the results of a two-year period of molecular diagnosis that included 207 French families referred for non-syndromic hearing loss. Our multi-step strategy involved (i) DFNB1 locus analysis, (ii) MPS of 74 genes, and (iii) additional approaches including Copy Number Variations, in silico analyses, minigene studies coupled when appropriate with complete gene sequencing, and a specific assay for STRC. This comprehensive screening yielded an overall diagnostic rate of 48%, equally distributed between DFNB1 (24%) and the other genes (24%). Pathogenic genotypes were identified in 19 different genes, with a high prevalence of GJB2, STRC, MYO15A, OTOF, TMC1, MYO7A and USH2A. Involvement of an Usher gene was reported in 16% of the genotyped cohort. Four de novo variants were identified. This study highlights the need to develop several molecular approaches for efficient molecular diagnosis of hearing loss, as this is crucial for genetic counselling, audiological rehabilitation and the detection of syndromic forms.

Most of the 2,000 variants identified in the CFTR (cystic fibrosis transmembrane regulator) gene are rare or private. Their interpretation is hampered by the lack of available data and resources, making patient care and genetic counseling challenging. We developed a patient-based database dedicated to the annotations of rare CFTR variants in the context of their cis- and trans-allelic combinations. Based on almost 30 years of experience of CFTR testing, CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr) currently compiles 16,819 variant records from 4,615 individuals with cystic fibrosis (CF) or CFTR-RD (related disorders), fetuses with ultrasound bowel anomalies, newborns awaiting clinical diagnosis, and asymptomatic compound heterozygotes. For each of the 736 different variants reported in the database, patient characteristics and genetic information (other variations in cis or in trans) have been thoroughly checked by a dedicated curator. Combining updated clinical, epidemiological, in silico, or in vitro functional data helps to the interpretation of unclassified and the reassessment of misclassified variants. This comprehensive CFTR database is now an invaluable tool for diagnostic laboratories gathering information on rare variants, especially in the context of genetic counseling, prenatal and preimplantation genetic diagnosis. CFTR-France is thus highly complementary to the international database CFTR2 focused so far on the most common CF-causing alleles., (© 2017 Wiley Periodicals, Inc.)

Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression controlled by multiple post- and co-transcriptional mechanisms. The large Duchenne muscular dystrophy gene encoding the protein dystrophin provides a striking example of the complexity of human pre-mRNAs. In this review, we summarize the current state of knowledge about canonical and non-canonical splicing in the DMD pre-mRNA, with a focus on mechanisms that take place in the full-length transcript isoform expressed in human skeletal muscle. In particular, we highlight recent work demonstrating that multi-step events are required for long DMD intron removal. The role of temporary intron retention in the occurrence of alternative splicing events is also discussed. Even though the proportion of splicing mutations is lower than reported in other genes, a great diversity of splicing defects linked to point mutations, but also large genomic rearrangements are observed in the DMD gene. We provide an overview of the molecular mechanisms underlying aberrant splicing in patients with Duchenne or Becker muscular dystrophy, and we also detail how alternative splicing can serve as a disease modifier in patients by changing the outcome of the primary defect.

The development of suitable Cystic Fibrosis (CF) models for preclinical bench tests of therapeutic candidates is challenging. Indeed, the validation of molecules to rescue the p.Phe508del-CFTR channel (encoded by the Cystic Fibrosis Transmembrane conductance Regulator gene carrying the p.Phe508del mutation) requires taking into account their overall effects on the epithelium. Suberoylanilide Hydroxamic Acid (SAHA), a histone deacetylase inhibitor (HDACi), was previously shown to be a CFTR corrector via proteostasis modulation in CFTR-deficient immortalized cells. Here, we tested SAHA effects on goblet cell metaplasia using an ex vivo model based on the air-liquid interface (ALI) culture of differentiated airway epithelial cells obtained by nasal scraping from CF patients and healthy controls. Ex vivo epithelium grew successfully in ALI cultures with significant rise in the expression of CFTR and of markers of airway epithelial differentiation compared to monolayer cell culture. SAHA decreased CFTR transcript and protein levels in CF and non-CF epithelia. Whereas SAHA induced lysine hyperacetylation, it did not change histone modifications at the CFTR promoter. SAHA reduced MUC5AC and MUC5B expression and inhibited goblet epithelial cell differentiation. Similar effects were obtained in CF and non-CF epithelia. All the effects were fully reversible within five days from SAHA withdrawal. We conclude that, ex vivo, SAHA modulate the structure of airway epithelia without specific effect on CFTR gene and protein suggesting that HDACi cannot be useful for CF treatment., (Copyright © 2017. Published by Elsevier Ltd.)

Background: Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches., Methods: To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence., Results: We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses., Conclusions: This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF., (Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: Lung disease progression is variable among cystic fibrosis (CF) patients and depends on DNA mutations in the CFTR gene, polymorphic variations in disease modifier genes, and environmental exposure. The contribution of genetic factors has been extensively investigated, whereas the mechanism whereby environmental factors modulate the lung disease is unknown. In this project, we hypothesized that (i) reiterative stress alters the epigenome in CF-affected tissues and (ii) DNA methylation variations at disease modifier genes modulate the lung function in CF patients., Results: We profiled DNA methylation at CFTR , the disease-causing gene, and at 13 lung modifier genes in nasal epithelial cells and whole blood samples from 48 CF patients and 24 healthy controls. CF patients homozygous for the p.Phe508del mutation and ≥18-year-old were stratified according to the lung disease severity. DNA methylation was measured by bisulfite and next-generation sequencing. The DNA methylation profile allowed us to correctly classify 75% of the subjects, thus providing a CF-specific molecular signature. Moreover, in CF patients, DNA methylation at specific genes was highly correlated in the same tissue sample. We suggest that gene methylation in CF cells may be co-regulated by disease-specific trans -factors. Three genes were differentially methylated in CF patients compared with controls and/or in groups of pulmonary severity: HMOX1 and GSTM3 in nasal epithelial samples; HMOX1 and EDNRA in blood samples. The association between pulmonary severity and DNA methylation at EDNRA was confirmed in blood samples from an independent set of CF patients. Also, lower DNA methylation levels at GSTM3 were associated with the GSTM3*B allele, a polymorphic 3-bp deletion that has a protective effect in cystic fibrosis., Conclusions: DNA methylation levels are altered in nasal epithelial and blood cell samples from CF patients. Analysis of CFTR and 13 lung disease modifier genes shows DNA methylation changes of small magnitude: some of them are a consequence of the disease; other changes may result in small expression variations that collectively modulate the lung disease severity.

This manuscript presents a molecularly demonstrated gonadal mosaicism from paternal origin for X-linked dominant chondrodysplasia punctata by single sperm typing. A couple who had experienced two medical terminations of pregnancy of female fetuses was referred to our pre-implantation genetic diagnosis (PGD) centre with the diagnosis of maternally derived gonadal mosaicism. Indeed, genetic analyses of different DNA samples - including semen - from the healthy parents failed to detect the variant found in the fetuses. Six embryos, all male, were obtained during the PGD cycle. The causative variant was not detected in any embryo, whereas five embryos had inherited the 'at-risk' maternal haplotype. The assumption of a maternal gonadal mosaicism was still possible, but this finding allowed us to consider the possibility of a paternal rather than maternal gonadal mosaicism. It prompted us to perform extensive single sperm analyses, demonstrating a low-frequency paternal germline mosaicism, which led to completely different haplotype phasing and PGD counselling. In conclusion, this case further exemplifies that germline mosaicism is a pitfall in PGD where diagnosis largely relies on linkage analysis and suggests that tracing the parental inheritance through polar body analysis and/or single sperm typing experiments is of major importance for adequate genetic counselling and accurate PGD. © 2016 John Wiley & Sons, Ltd., (© 2016 John Wiley & Sons, Ltd.)

We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3' splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.

Next-generation sequencing (NGS) has an established diagnostic value for inherited ataxia. However, the need of a rigorous process of analysis and validation remains challenging. Moreover, copy number variations (CNV) or dynamic expansions of repeated sequence are classically considered not adequately detected by exome sequencing technique. We applied a strategy of mini-exome coupled to read-depth based CNV analysis to a series of 33 patients with probable inherited ataxia and onset <50 years. The mini-exome consisted of the capture of 4,813 genes having associated clinical phenotypes. Pathogenic variants were found in 42% and variants of uncertain significance in 24% of the patients. These results are comparable to those from whole exome sequencing and better than previous targeted NGS studies. CNV and dynamic expansions of repeated CAG sequence were identified in three patients. We identified both atypical presentation of known ataxia genes (ATM, NPC1) and mutations in genes very rarely associated with ataxia (ERCC4, HSD17B4). We show that mini-exome bioinformatics data analysis allows the identification of CNV and dynamic expansions of repeated sequence. Our study confirms the diagnostic value of the proposed genetic analysis strategy. We also provide an algorithm for the multidisciplinary process of analysis, interpretation, and validation of NGS data., (© 2016 WILEY PERIODICALS, INC.)

The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor β [TGFβ]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8 × 10 -6 ). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFβ pathways. The minor allele at rs1883832, in the 5'-untranslated region of CD40, was associated with earlier LoA (p = 3.5 × 10 -5 ). This allele diminishes the expression of CD40, a co-stimulatory molecule for T cell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD., (Copyright © 2016. Published by Elsevier Inc.)

Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.

Diagnosis of dystrophinopathies needs to combine several techniques for detecting copy number variations (CNVs; two-thirds of mutations) and single nucleotide variations (SNVs). We participated in the design of an amplicon-based PCR kit (Multiplicom) for sequencing with a GS-Junior instrument (Roche) and later with a MiSeq instrument (Illumina). We compared two different software programs, MiSeq Reporter (Illumina) and SeqNext (JSI Medical Systems) for data analyses. Testing of six patient DNA samples carrying 72 SNVs in the DMD gene showed an experimental sensitivity of 91.7% with MiSeq Reporter, 98.6% with SeqNext, and >99.9% with both, demonstrating the need to use two different software programs. Analytical specificity was >98%. Fifty-eight additional patient DNAs were analyzed, and 25 deleterious mutations were identified, without false-negative results. We also tested the possibility for our protocol to identify CNVs. We performed additional next-generation sequencing experiments on 50 DNAs and identified 28 CNVs, all confirmed by multiple ligation probe amplification. Statistical analyses on amplicons without CNV (n = 3797), amplicons with heterozygous deletions (n = 51) or duplications (n = 191), and with hemizygous duplications (n = 63) showed a sensitivity and specificity of >99.9%. We implemented a strategy to simultaneously detect SNVs and CNVs in the DMD gene with one comprehensive technique, allowing considerable reduction of time and cost burden for diagnosis of dystrophinopathies., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Peroxisomal biogenesis disorders (PBDs) consist of a heterogeneous group of autosomal recessive diseases, in which peroxisome assembly and proliferation are impaired leading to severe multisystem disease and early death. PBDs include Zellweger spectrum disorders (ZSDs) with a relatively mild clinical phenotype caused by PEX1, (MIM# 602136), PEX2 (MIM# 170993), PEX6 (MIM# 601498), PEX10 (MIM# 602859), PEX12 (MIM# 601758), and PEX16 (MIM# 603360) mutations. Three adult patients are reported belonging to a non-consanguineous French family affected with slowly progressive cerebellar ataxia, axonal neuropathy, and pyramidal signs. Mental retardation and diabetes mellitus were optional. The age at onset was in childhood or in adolescence (3-15 years). Brain MRI showed marked cerebellar atrophy. Biochemical blood analyses suggested a mild peroxisomal defect. With whole exome sequencing, two mutations in PEX10 were found in the three patients: c.827G>T (novel) causing the missense change p.Cys276Phe and c.932G>A causing the missense change p.Arg311Gln. The phenotypic spectrum related to PEX10 mutations includes slowly progressive, syndromic recessive ataxia.

Ataxia is a symptom that is often associated with syndromic inherited diseases. We previously reported the linkage of a novel syndrome, ataxia with blindness and deafness (SCAR3/SCABD, OMIM# 271250), to chromosome 6p21-p23 by linkage mapping of an Arab Israeli consanguineous family. We have now identified by whole-exome sequencing a homozygous missense mutation in the Arab Israeli family in the SLC52A2 gene located in 8qter, therefore excluding linkage of this family to 6p. We confirmed the involvement of SLC52A2 by the identification of a second mutation in an independent family with an identical syndromic presentation, which we suggest to name SCABD2. SCABD2 is therefore allelic to Brown-Vialleto-Van Laere syndrome type 2 defined by prominent motoneuronopathy and deafness, and also caused by SLC52A2 mutations. In the course of this project, we identified a clinically similar family with a homozygous missense mutation in PEX6, which is located in 6p21. Therefore, despite false linkage in the initial family, SCABD1/SCAR3 is located in 6p21 and is caused by PEX6 mutations. Both SLC52A2 and PEX6 should be included in screening panels for the diagnosis of syndromic inherited ataxias, particularly as patients with mutations in SLC52A2 can be ameliorated by riboflavin supplementation.

Background: Dystrophinopathies are mostly caused by copy number variations, especially deletions, in the dystrophin gene (DMD). Despite the large size of the gene, deletions do not occur randomly but mainly in two hot spots, the main one involving exons 45 to 55. The underlying mechanisms are complex and implicate two main mechanisms: Non-homologous end joining (NHEJ) and micro-homology mediated replication-dependent recombination (MMRDR)., Objective: Our goals were to assess the distribution of intronic breakpoints (BPs) in the genomic sequence of the main hot spot of deletions within DMD gene and to search for specific sequences at or near to BPs that might promote BP occurrence or be associated with DNA break repair., Methods: Using comparative genomic hybridization microarray, 57 deletions within the intron 44 to 55 region were mapped. Moreover, 21 junction fragments were sequenced to search for specific sequences., Results: Non-randomly distributed BPs were found in introns 44, 47, 48, 49 and 53 and 50% of BPs clustered within genomic regions of less than 700bp. Repeated elements (REs), known to promote gene rearrangement via several mechanisms, were present in the vicinity of 90% of clustered BPs and less frequently (72%) close to scattered BPs, illustrating the important role of such elements in the occurrence of DMD deletions. Palindromic and TTTAAA sequences, which also promote DNA instability, were identified at fragment junctions in 20% and 5% of cases, respectively. Micro-homologies (76%) and insertions or deletions of small sequences were frequently found at BP junctions., Conclusions: Our results illustrate, in a large series of patients, the important role of RE and other genomic features in DNA breaks, and the involvement of different mechanisms in DMD gene deletions: Mainly replication error repair mechanisms, but also NHEJ and potentially aberrant firing of replication origins. A combination of these mechanisms may also be possible.

The detection of two frequent CFTR disease-causing variations in the context of a newborn screening program (NBS) usually leads to the diagnosis of cystic fibrosis (CF) and a relevant genetic counseling in the family. In the present study, CF-causing variants p.Phe508del (F508del) and c.3140-26A>G (3272-26A>G) were identified on a neonate with positive ImmunoReactive Trypsinogen test by the Elucigene™ CF30 kit. The CF diagnosis initially suggested, despite three inconclusive Sweat Chloride Tests (SCT), was finally ruled out after the familial segregation study combined with a negative SCT. Haplotype studies, based on the comparison of 80 p.Phe508del haplotypes, suggested a probable de novo occurrence of c.3140-26A>G on the p.Phe508del ancestral allele in this family. This false positive case emphasizes the importance of SCT in the NBS strategy. Moreover, it raises the need for familial segregation studies in CF and in overall molecular diagnosis strategy of autosomal recessive diseases., (Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented.

Enrichment capture methods for NGS are widely used, however, they evolve rapidly and it is necessary to periodically measure their strengths and weaknesses before transfer to diagnostic services. We assessed two recently released custom DNA solution-capture enrichment methods for NGS, namely Illumina NRCCE and Agilent SureSelect(QXT), against a reference method NimbleGen SeqCap EZ Choice on a similar gene panel, sharing 678 kb and 110 genes. Two Illumina MiSeq runs of 12 samples each have been performed, for each of the three methods, using the same 24 patients (affected with sensorineural disorders). Technical outcomes have been computed and compared, including depth and evenness of coverage, enrichment in targeted regions, performance in GC-rich regions and ability to generate consistent variant datasets. While we show that the three methods resulted in suitable datasets for standard DNA variant discovery, we describe significant differences between the results for the above parameters. NimbleGen offered the best depth of coverage and evenness, while NRCCE showed the highest on target levels but high duplicate rates. SureSelect(QXT) showed an overall quality close to that of NimbleGen. The new methods exhibit reduced preparation time but behave differently. These findings will guide laboratories in their choice of library enrichment approach.

Deep intronic mutations leading to pseudoexon (PE) insertions are underestimated and most of these splicing alterations have been identified by transcript analysis, for instance, the first deep intronic mutation in USH2A, the gene most frequently involved in Usher syndrome type II (USH2). Unfortunately, analyzing USH2A transcripts is challenging and for 1.8%-19% of USH2 individuals carrying a single USH2A recessive mutation, a second mutation is yet to be identified. We have developed and validated a DNA next-generation sequencing approach to identify deep intronic variants in USH2A and evaluated their consequences on splicing. Three distinct novel deep intronic mutations have been identified. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. We present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. Moreover, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Finally, an antisense morpholino oligonucleotide tested in vitro for its ability to restore splicing caused by the c.9959-4159A>G mutation provided high inhibition rates, which are indicative of its potential for molecular therapy., (© 2015 WILEY PERIODICALS, INC.)

In 2013, the American College of Medical Genetics (ACMG) examined the issue of incidental findings in whole exome and whole genome sequencing, and introduced recommendations to search for, evaluate and report medically actionable variants in a set of 56 genes. At a debate held during the 2014 European Society for Human Genetics Conference (ESHG) in Milan, Italy, the first author of that paper presented this view in a debate session that did not end with a conclusive vote from the mainly European audience for or against reporting back actionable incidental findings. In this meeting report, we elaborate on the discussions held during a special meeting hosted at the ESHG in 2013 from posing the question 'How to reach a (European) consensus on reporting incidental findings and unclassified variants in diagnostic next generation sequencing'. We ask whether an European consensus exists on the reporting of incidental findings in genome diagnostics, and present a series of key issues that require discussion at both a national and European level in order to develop recommendations for handling incidental findings and unclassified variants in line with the legal and cultural particularities of individual European member states.

Objective: Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials., Methods: Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates., Results: We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss., Conclusions: This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Purpose: Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations., Methods: We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified., Results: Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF., Conclusion: Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.

Background: Although several comprehensive studies have evaluated the role of the CFTR gene in idiopathic diffuse bronchiectasis (DB), it remains controversial., Methods: We analyzed the whole coding region of the CFTR gene, its flanking regions and the promoter in 47 DB patients and 47 controls. Available information about demographic, spirometric, radiological and microbiological data for the DB patients was collected. Unclassified CFTR variants were in vitro functionally assessed., Results: CFTR variants were identified in 24 DB patients and in 27 controls. DB variants were reclassified based on the results of in silico predictive analyses, in vitro functional assays and data from epidemiological and literature databases. Except for the sweat test value, no clear genotype-phenotype correlation was observed., Conclusions: DB should not be considered a classical autosomal recessive CFTR-RD. Moreover, although further investigations are necessary, we proposed a new class of "Non-Neutral Variants" whose impact on lung disease requires more studies., (Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Defects in TRIM32 were reported in limb-girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathies (STM) and in Bardet-Biedl syndrome. Few cases have been described to date in LGMD2H/STM, but this gene is not systematically analysed because of the absence of specific signs and difficulties in protein analysis. By using high-throughput variants screening techniques, we identified variants in TRIM32 in two patients presenting nonspecific LGMD. We report the first case of total inactivation by homozygous deletion of the entire TRIM32 gene. Of interest, the deletion removes part of the ASTN2 gene, a large gene in which TRIM32 is nested. Despite the total TRIM32 gene inactivation, the patient does not present a more severe phenotype. However, he developed a mild progressive cognitive impairment that may be related to the loss of function of ASTN2 because association between ASTN2 heterozygous deletions and neurobehavioral disorders was previously reported. Regarding genomic characteristics at breakpoint of the deleted regions of TRIM32, we found a high density of repeated elements, suggesting a possible hotspot. These observations illustrate the importance of high-throughput technologies for identifying molecular defects in LGMD, confirm that total loss of function of TRIM32 is not associated with a specific phenotype and that TRIM32/ASTN2 inactivation could be associated with cognitive impairment.