The FLNC Ala1186Val Variant Linked to Cytoplasmic Body Myopathy and Cardiomyopathy Causes Protein Instability.

Filamin C-related disorders include myopathies and cardiomyopathies linked to variants in the FLNC gene. Filamin C belongs to a family of actin-binding proteins involved in sarcomere stability. This study investigates the pathogenic impact of the FLNC c.3557C > T (p.Ala1186Val) pathogenic variant associated with an early-onset cytoplasmic body myopathy and cardiomyopathy in three unrelated patients. We performed clinical imaging and myopathologic and genetic characterization of three patients with an early-onset myopathy and cardiomyopathy. Bioinformatics analysis, variant interpretation, and protein structure analysis were performed to validate and assess the effects of the filamin C variant. All patients presented with a homogeneous clinical phenotype marked by a severe contractural myopathy, leading to loss of gait. There was prominent respiratory involvement and restrictive or hypertrophic cardiomyopathies. The Ala1186Val variant is located in the interstrand loop involved in intradomain stabilization and/or interdomain interactions with neighbor Ig-like domains. 3D modeling highlights local structural changes involving nearby residues and probably impacts the protein stability, causing protein aggregation in the form of cytoplasmic bodies. Myopathologic studies have disclosed the prominent aggregation and upregulation of the aggrephagy-associated proteins LC3B and p62. As a whole, the Ala1186Val variant in the FLNC gene provokes a severe myopathy with contractures, respiratory involvement, and cardiomyopathy due to protein aggregation in patients' muscles.

Evaluating the Transition from Targeted to Exome Sequencing: A Guide for Clinical Laboratories.

The transition from targeted to exome or genome sequencing in clinical contexts requires quality standards, such as targeted sequencing, in order to be fully adopted. However, no clear recommendations or methodology have emerged for evaluating this technological evolution. We developed a structured method based on four run-specific sequencing metrics and seven sample-specific sequencing metrics for evaluating the performance of exome sequencing strategies to replace targeted strategies. The indicators include quality metrics and coverage performance on gene panels and OMIM morbid genes. We applied this general strategy to three different exome kits and compared them with a myopathy-targeted sequencing method. After having achieved 80 million reads, all-tested exome kits generated data suitable for clinical diagnosis. However, significant differences in the coverage and PCR duplicates were observed between the kits. These are two main criteria to consider for the initial implementation with high-quality assurance. This study aims to assist molecular diagnostic laboratories in adopting and evaluating exome sequencing kits in a diagnostic context compared to the strategy used previously. A similar strategy could be used to implement whole-genome sequencing for diagnostic purposes.

Long-Reads Sequencing Strategy to Localize Variants in TTN Repeated Domains.

Titin protein is responsible for muscle elasticity. The TTN gene, composed of 364 exons, is subjected to extensive alternative splicing and leads to different isoforms expressed in skeletal and cardiac muscle. Variants in TTN are responsible for myopathies with a wide phenotypic spectrum and autosomal dominant or recessive transmission. The I-band coding domain, highly subject to alternative splicing, contains a three-zone block of repeated sequences with 99% homology. Sequencing and localization of variants in these areas are complex when using short-reads sequencing, a second-generation sequencing technique. We have implemented a protocol based on the third-generation sequencing technology (long-reads sequencing). This new method allows us to localize variants in these repeated areas to improve the diagnosis of TTN-related myopathies and offer the analysis of relatives in postnatal or in prenatal screening.

An Integrated Clinical-Biological Approach to Identify Interindividual Variability and Atypical Phenotype-Genotype Correlations in Myopathies: Experience on A Cohort of 156 Families.

Diagnosis of myopathies is challenged by the high genetic heterogeneity and clinical overlap of the various etiologies. We previously reported a Next-Generation Sequencing strategy to identify genetic etiology in patients with undiagnosed Limb-Girdle Muscular Dystrophies, Congenital Myopathies, Congenital Muscular Dystrophies, Distal Myopathies, Myofibrillar Myopathies, and hyperCKemia or effort intolerance, using a large gene panel including genes classically associated with other entry diagnostic categories. In this study, we report the comprehensive clinical-biological strategy used to interpret NGS data in a cohort of 156 pediatric and adult patients, that included Copy Number Variants search, variants filtering and interpretation according to ACMG guidelines, segregation studies, deep phenotyping of patients and relatives, transcripts and protein studies, and multidisciplinary meetings. Genetic etiology was identified in 74 patients, a diagnostic yield (47.4%) similar to previous studies. We identified 18 patients (10%) with causative variants in different genes (ACTA1, RYR1, NEB, TTN, TRIP4, CACNA1S, FLNC, TNNT1, and PAPBN1) that resulted in milder and/or atypical phenotypes, with high intrafamilial variability in some cases. Mild phenotypes could mostly be explained by a less deleterious effect of variants on the protein. Detection of inter-individual variability and atypical phenotype-genotype associations is essential for precision medicine, patient care, and to progress in the understanding of the molecular mechanisms of myopathies.

Novel dominant distal titinopathy phenotype associated with copy number variation.

The aim of this study was to analyze patients from two distinct families with a novel distal titinopathy phenotype associated with exactly the same CNV in the TTN gene. We used an integrated strategy combining deep phenotyping and complete molecular analyses in patients. The CNV is the most proximal out-of-frame TTN variant reported and leads to aberrant splicing transcripts leading to a frameshift. In this case, the dominant effect would be due to dominant-negative and/or haploinsufficiency. Few CNV in TTN have been reported to date. Our data represent a novel phenotype-genotype association and provides hypotheses for its dominant effects.

Evaluating next-generation sequencing in neuromuscular diseases with neonatal respiratory distress.

With the exception of infantile spinal muscular atrophy (SMA) and congenital myotonic dystrophy 1 (DM1), congenital myopathies and muscular dystrophies with neonatal respiratory distress pose diagnostic challenges. Next-generation sequencing (NGS) provides hope for the diagnosis of these rare diseases. We evaluated the efficiency of next-generation sequencing (NGS) in ventilated newborns with peripheral hypotonia. We compared the results of our previous study in a cohort of 19 patients analysed by Sanger sequencing from 2007 to 2012, with a diagnostic yield of 26% (5/19), and those of a new retrospective study in 28 patients from 2007 to 2018 diagnosed using MyoPanel, a neuromuscular disease panel, with a diagnostic yield of 43% (12/28 patients). Pathogenic variants were found in five genes: ACTA1 (n = 4 patients), RYR1 (n = 2), CACNA1S (n = 1), NEB (n = 3), and MTM1 (n = 2). Myopanel increased the diagnosis of congenital neuromuscular diseases, but more than half the patients remained undiagnosed. Whole exome sequencing did not seem to fully respond to this diagnostic limitation. Therefore, explorations with whole genome sequencing will be the next step.

Clinical phenotype and loss of the slow skeletal muscle troponin T in three new patients with recessive TNNT1 nemaline myopathy.

BACKGROUND: Congenital nemaline myopathies are rare pathologies characterised by muscle weakness and rod-shaped inclusions in the muscle fibres. METHODS: Using next-generation sequencing, we identified three patients with pathogenic variants in the Troponin T type 1 (TNNT1) gene, coding for the troponin T (TNT) skeletal muscle isoform. RESULTS: The clinical phenotype was similar in all patients, associating hypotonia, orthopaedic deformities and progressive chronic respiratory failure, leading to early death. The anatomopathological phenotype was characterised by a disproportion in the muscle fibre size, endomysial fibrosis and nemaline rods. Molecular analyses of TNNT1 revealed a homozygous deletion of exons 8 and 9 in patient 1; a heterozygous nonsense mutation in exon 9 and retention of part of intron 4 in muscle transcripts in patient 2; and a homozygous, very early nonsense mutation in patient 3.Western blot analyses confirmed the absence of the TNT protein resulting from these mutations. DISCUSSION: The clinical and anatomopathological presentations of our patients reinforce the homogeneous character of the phenotype associated with recessive TNNT1 mutations. Previous studies revealed an impact of recessive variants on the tropomyosin-binding affinity of TNT. We report in our patients a complete loss of TNT protein due to open reading frame disruption or to post-translational degradation of TNT.

The importance of an integrated genotype-phenotype strategy to unravel the molecular bases of titinopathies.

Next generation sequencing (NGS) has allowed the titin gene (TTN) to be identified as a major contributor to neuromuscular disorders, with high clinical heterogeneity. The mechanisms underlying the phenotypic variability and the dominant or recessive pattern of inheritance are unclear. Titin is involved in the formation and stability of the sarcomeres. The effects of the different TTN variants can be harmless or pathogenic (recessive or dominant) but the interpretation is tricky because the current bioinformatics tools can not predict their functional impact effectively. Moreover, TTN variants are very frequent in the general population. The combination of deep phenotyping associated with RNA molecular analyses, western blot (WB) and functional studies is often essential for the interpretation of genetic variants in patients suspected of titinopathy. In line with the current guidelines and suggestions, we implemented for patients with skeletal myopathy and with potentially disease causing TTN variant(s) an integrated genotype-transcripts-protein-phenotype approach, associated with phenotype and variants segregation studies in relatives and confrontation with published data on titinopathies to evaluate pathogenic effects of TTN variants (even truncating ones) on titin transcripts, amount, size and functionality. We illustrate this integrated approach in four patients with recessive congenital myopathy.

CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants.

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.

Small-scale high-throughput sequencing-based identification of new therapeutic tools in cystic fibrosis.

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.

A classification model relative to splicing for variants of unknown clinical significance: application to the CFTR gene.

Molecular diagnosis of cystic fibrosis and cystic fibrosis transmembrane regulator (CFTR)-related disorders led to the worldwide identification of nearly 1,900 sequence variations in the CFTR gene that consist mainly of private point mutations and small insertions/deletions. Establishing their effect on the function of the encoded protein and therefore their involvement in the disease is still challenging and directly impacts genetic counseling. In this context, we built a decision tree following the international guidelines for the classification of variants of unknown clinical significance (VUCS) in the CFTR gene specifically focused on their consequences on splicing. We applied general and specific criteria, including comprehensive review of literature and databases, familial genetics data, and thorough in silico studies. This model was tested on 15 intronic and exonic VUCS identified in our cohort. Six variants were classified as probably nonpathogenic considering their impact on splicing and eight as probably pathogenic, which include two apparent missense mutations. We assessed the validity of our method by performing minigenes studies and confirmed that 93% (14/15) were correctly classified. We provide in this study a high-performance method that can play a full role in interpreting the results of molecular diagnosis in emergency context, when functional studies are not achievable.

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene.

In European populations, large rearrangements contribute to approximately 2% of CF mutations. Here, we reported a novel duplication, the CFTRdup2, identified in a patient heterozygous for Phe508del and suffering from a mild CF. Using a combination of functional tests, we studied the impact of duplication/deletion on CFTR expression. We showed that the copy number variations of exon 2, in addition to abolishing the rate of the mature CFTR protein, affect the CFTR mRNA levels. These data illustrate the importance to perform functional analysis to better understand the molecular basis responsible for cystic fibrosis. Determining the impact of deletions or duplications is relevant for a more comprehensive diagnosis and prognosis of patients.

UMD-CFTR: a database dedicated to CF and CFTR-related disorders.

With the increasing knowledge of cystic fibrosis (CF) and CFTR-related diseases (CFTR-RD), the number of sequence variations in the CFTR gene is constantly raising. CF and particularly CFTR-RD provide a particular challenge because of many unclassified variants and identical genotypes associated with different phenotypes. Using the Universal Mutation Database (UMD) software we have constructed UMD-CFTR (freely available at the URL: http://www.umd.be/CFTR/), the first comprehensive relational CFTR database that allows an in-depth analysis and annotation of all variations identified in individuals whose CFTR genes have been analyzed extensively. The system has been tested on the molecular data from 757 patients (540 CF and 217 CBAVD) including disease-causing, unclassified, and nonpathogenic alterations (301 different sequence variations) representing 3,973 entries. Tools are provided to assess the pathogenicity of mutations. UMD-CFTR also offers a number of query tools and graphical views providing instant access to the list of mutations, their frequencies, positions and predicted consequences, or correlations between genotypes, haplotypes, and phenotypes. UMD-CFTR offers a way to compile not only disease-causing genotypes but also haplotypes. It will help the CFTR scientific and medical communities to improve sequence variation interpretation, evaluate the putative influence of haplotypes on mutations, and correlate molecular data with phenotypes.

A novel double deletion underscores the importance of characterizing end points of the CFTR large rearrangements.

Large genomic rearrangements in patients with cystic fibrosis (CF) account for up to 16-24% of CF alleles negative for point mutations in European populations. Herein, we identified a new large rearrangement removing exon 19 in a young CF patient, who hitherto harbored only the F508del mutation. By using LightCycler technology, we successfully and rapidly delineated the deletion end points by determining the relative copy number of a set CFTR sequence from introns 18 to 19. Fine mapping of the sequences bordering its break points was achieved using direct sequencing. We reported the first complex CFTR rearrangement containing two successive deletion events putatively linked. We evidenced the presence of short direct repeats in the vicinity of the deletions suggesting a possible replication slippage model. In this report, we also discussed the putative molecular mechanism and consequences of this complex gene rearrangement, unprecedented in CF. This complex deletion illustrates the importance of delineating the genomic rearrangement to improve our knowledge of the CFTR mutational spectrum and to better understand the molecular mechanism controlling the CFTR expression.