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Single-gene copy number variants (CNVs) limited to placenta although rarely identified may have clinical implications. We describe a pregnant woman referred for chorionic villus sampling due to increased fetal nuchal translucency. Incident intragenic deletion of Duchenne muscular dystrophy (DMD) gene, affecting exons 56 and 57, was identified in a male fetus in ~23-30% of placental cells by chromosomal microarray and confirmed using multiplex ligation-dependent probe amplification (MLPA). Rapid aneuploidy testing showed normal results and the deletion was not detected in the mother. Subsequent analyses on amniotic cells yielded a normal DMD gene result, corroborating the confined placental nature of the mosaicism. Hence, this report emphasizes the importance of conducting amniocentesis following detection of mosaicism for single gene CNVs on chorionic villi, in order to preclude confined placental mosaicism (CPM). As far as we know, this report marks only the second documented situation of CPM involving an intragenic DMD deletion., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

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.)

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., (© 2013 Wiley Periodicals, Inc.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.