High-throughput genome and exome sequencing have uncovered numerous intronic variants in disease genes, yet predicting their impact on pre-mRNA splicing is still challenging. While deep learning tools have improved variant prioritization, their ability to detect atypical regulatory mechanisms remains unknown. We combined predictions from multiple complementary splicing prediction tools with functional RNA studies to evaluate the splicing consequences of intronic variants identified in patients with neurodegenerative diseases. Nine intronic variants with uncertain impact on splicing were selected, including six deep-intronic variants, two non-canonical splice-site variants, and one canonical splice-site variant associated with an atypical phenotype. Three variants were predicted to disrupt a donor splice site, five to create a cryptic donor splice site, and one was located close to a putative acceptor site. Transcript analysis in blood revealed splicing abnormalities in eight of the nine cases. All algorithms failed at least once: deep learning tools missed specific donor site losses or cryptic splice-site activation events, while motif-based approaches failed to predict some donor site activation or generated false-positive predictions. The only variant not directly located in a splice site was predicted to activate a deep intronic splicing enhancer. Motif-based tools suggested the creation of an SRSF2 protein-dependent enhancer. Functional studies based on minigene assays support a role for SRSF2 in promoting pseudoexon inclusion. These findings highlight the complementarity of splicing prediction tools and the need to integrate them into diagnostic pipelines, with transcript-level confirmation remaining critical for accurate pathogenicity assessment.

Titinopathies are complex neuromuscular disorders with multiple phenotypes. The gene's size, comprising 364 exons, as well as the protein's size of 3.8 MDa and its extensive network of protein interactors, are key factors underlying this complexity. Various phenotypes characterize titinopathies, and this study focuses on two of them: arthrogryposis and myofibrillar myopathies. The protein deregulations associated with these two phenotypes remain unknown or have been minimally explored; however, understanding these consequences is essential for better characterizing the pathophysiological aspects of these titinopathies.The objective was to analyze protein deregulations in two cohorts of French patients with titinopathies exhibiting the arthrogryposis and myofibrillar myopathy phenotypes, and to compare them with control individuals. Protein extracts were obtained from muscle biopsies of patients, and changes in protein levels within these two groups were analyzed by mass spectrometry. The results indicate specific deregulations in each group. The networks analyzed revealed deregulation of proteins involved in fibrosis mechanisms or in the actomyosin complex for the arthrogryposis phenotype. Regulation of the muscle contraction system through deregulation of proteins involved in the cytoskeleton is impacted in patients with myofibrillar myopathy. The proteins that are quantitatively abnormal in these two groups also provide insights into the major signaling networks disrupted in titinopathies. These findings will contribute to a more precise characterization of titinopathies, enabling the identification of phenotype-specific biomarkers and potentially guiding the search for targeted therapies for these neuromuscular disorders.

Introduction SCA27A est une ataxie dominante liée à des variants conventionnels dans FGF14 [1]. SCA27B est une ataxie dominante liée à une expansion GAA intronique dans le même gène [2]. Objectifs Mieux caractériser le phénotype de SCA27A, et le comparer avec celui de SCA27B. Une meilleure définition phénotypique pourrait permettre d’élaborer des hypothèses sur les mécanismes pathogéniques. Méthodes SCA27A : étude rétrospective internationale multicentrique (consortium SCANOP et SCA27B). SCA27B : recueil des données phénotypiques à partir des séries récentes publiées [3], [4]. Résultats Un un total de 41 patients (28 familles) avec SCA27A avec début moyen à 13 ans. Le plus fréquent symptôme initial est l’ataxie, avec différentes manifestations (oscillopsie, tremblement, déséquilibre) en fonction de l’âge de début. Une déficience intellectuelle ou des troubles de l’apprentissage sont décrits dans 27/41 patients. Le suivi moyen est 21 ans avec un score SARA de 10.6/40. On retrouve des symptômes paroxystiques avec ataxie (n=14), oscillopsie (n=3), migraines (n=9), épilepsie(n=5), dystonie(n=3). La 4-dyaminopiridine a été essayée dans 6 patients, avec une amélioration dans deux. Au niveau génétique nous retrouvons des délétions 13q33.1 incluant FGF14 (n=19), 7 variants non-sens et 2 faux-sens. Discussion SCA27A, comme SCA27B, est une ataxie avec évolution lente et des symptômes épisodiques et oculomoteurs. La réponse à la 4-dyaminopiridine a été rarement testée dans SCA27A. Les principales différences sont l’âge de début moyenne (13 ans pour SCA27A vs 45-60 ans pour SCA27B) et la composante développementale avec déficience intellectuelle. Conclusion Dans les deux cas, il s’agit de mutations induisant une haploinsuffisance. Pour SCA27A la perte de fonction est là dès le début, alors que pour SCA27B le mécanisme d’inhibition de l’expression de la protéine pourrait être plus tardif, préservant la période développementale.

PURPOSE:In Friedreich ataxia (FRDA) the size of the smaller GAA expansion is a major determinant of disease severity; interruption motifs were identified after the discovery of the pathogenic expansions; however, their impact is only recently investigated. METHODS:164 patients with FRDA with biallelic expansions and 15 patients without FRDA were analyzed for interruption(s) number, position, and motif. Expansion size and age at onset of ataxia (AAO) were determined for patients with FRDA. RESULTS:Three groups of patients with FRDA were identified by the simultaneous analysis of the precise distance ("depth") between the interruptions (mostly nontriplet) and the 3' end of the expansion (P < .001), the smaller expansion size (P < .001), and AAO (P < .001). Classical FRDA corresponds to absence of interruption or interruption depth < 8 repeats, with AAO often <15 years (area under the curve [AUC] = 0.90; 95% CI, 0.84-0.96); LOFA to interruption depth of 8 to 18 repeats (AUC = 0.97; 95% CI, 0.94-1), with AAO 15 to 34 years (AUC = 1; 95% CI, 1-1); and vLOFA to interruption depth > 18 (AUC = 0.97; 95% CI, 0.92-1), with AAO > 34 years. Multiple (>5) triplet interruptions hamper further expansion. CONCLUSION:This study provides the molecular basis for a novel classification of FRDA that should be recommended for correct diagnosis.

Atypical neuroaxonal dystrophy (ANAD) is a rare form of neurodegeneration linked to the PLA2G6 gene. Unlike classical infantile neuroaxonal dystrophy (INAD), it occurs later in childhood and seems less progressive. It appears phenotypically different from juvenile form of Parkinson disease linked to PLA2G6 (PARK14). A genotype-phenotype correlation has been suggested. We describe a large genetically confirmed cohort of pediatric patients with ANAD, describe their clinical symptomatology, brain imaging, other complementary explorations, symptomatic medication and compare to patients reported in the literature. Fourteen patients were identified with early childhood onset and slowly progressive cerebello-spastic syndrome with variable dystonia and parkinsonism. Complementary investigations were inconsistently abnormal compared to INAD, with variable iron deposits on brain imaging, infrequent rapid rhythms on EEG and absence of neuronal spheroids on skin biopsy leading to diagnosis difficulties in absence of large molecular analysis. Nine of the seventeen reported variants were novel variants and a relative genotype-phenotype correlation was confirmed. This study reports a large cohort of ANAD, providing new insights into this paediatric phenotype; which is less frequently described in the literature compared to INAD or PARK14.

OBJECTIVE:To report the incidental detection of maternal somatic mosaicism during the development of exclusion-based non-invasive prenatal diagnosis for monogenic disorders (NIPD-MD) initially indicated for apparently de novo pathogenic or likely pathogenic variants. METHOD:A droplet digital PCR (ddPCR)-based exclusion NIPD_MD assay was developed for four couples, each with a prior pregnancy affected by a rare autosomal dominant or X-linked condition due to a de novo pathogenic or likely pathogenic variant. Assays were designed to detect fetal-specific variants in maternal plasma, with validation performed on parental and proband samples. RESULTS:In four cases, maternal somatic mosaicism (3%-9%) among 70 personalized NIPD_MD (5.7%) was identified during assay validation, rendering NIPD_MD infeasible due to interference from maternal alleles. Each couple was informed of the elevated recurrence risk. Depending on their preferences, invasive prenatal testing or intensive ultrasound follow-up was undertaken. Retrospective analysis of maternal sequencing data confirmed low-level mosaicism that had been filtered out during routine analysis. CONCLUSION:These cases underscore a key limitation of exclusion NIPD_MD when maternal mosaicism is present. Its identification is essential for accurate recurrence risk estimation and genetic counseling. Sensitive detection methods, careful pre-test evaluation, and transparent communication are critical to ensure informed reproductive decision-making.

BACKGROUND:Titinopathies are caused by mutations in the titin gene (TTN). Titin is the largest known human protein; its gene has the longest coding phase with 364 exons. Titinopathies are very complex neuromuscular pathologies due to the variable age of onset of symptoms, the great diversity of pathological and muscular impairment patterns (cardiac, skeletal muscle or mixed) and both autosomal dominant and recessive modes of transmission. Until now, only few CNVs in TTN have been reported without clear genotype-phenotype associations. METHODS:Our study includes eight families with dominant titinopathies. We performed next-generation sequencing or comparative genomic hybridisation array analyses and found CNVs in the TTN gene. We characterised these CNVs by RNA sequencing (RNAseq) analyses in six patients' muscles and performed genotype-phenotype inheritance association study by combining the clinical and biological data of these eight families. RESULTS:Seven deletion-type CNVs in the TTN gene were identified among these families. Genotype and RNAseq results showed that five deletions do not alter the reading frame and one is out-of-reading frame. The main phenotype identified was distal myopathy associated with contractures. The analysis of morphological, clinical and genetic data and imaging let us draw new genotype-phenotype associations of titinopathies. CONCLUSION:Identifying TTN CNVs will further increase diagnostic sensitivity in these complex neuromuscular pathologies. Our cohort of patients enabled us to identify new deletion-type CNVs in the TTN gene, with unexpected autosomal dominant transmission. This is valuable in establishing new genotype-phenotype associations of titinopathies, mainly distal myopathy in most of the patients.

Congenital titinopathies reported to date show autosomal recessive inheritance and are caused by a variety of genomic variants, most of them located in metatranscript (MTT)-only exons. The aim of this study was to describe additional patients and establish robust genotype-phenotype associations in titinopathies. This study involved analyzing molecular, clinical, pathological, and muscle imaging features in 20 patients who had at least one pathogenic or likely pathogenic TTN variant in MTT-only exons, with onset occurring antenatally or in the early postnatal stages. The 20 patients with recessive inheritance exhibited a heterogeneous range of phenotypes. These included fetal lethality, progressive weakness, cardiac or respiratory complications, hyper-CKemia, or dystrophic muscle biopsies. MRI revealed variable abnormalities in different muscles. All patients presented severe congenital myopathy at birth, characterized by arthrogryposis (either multiplex or axial-distal) or neonatal hypotonia in most cases. This study provides detailed genotype-phenotype correlations in congenital titinopathies caused by mutations in MTT-only exons. The findings highlight the variability in clinical presentation and the severity of phenotypes associated with these specific genetic alterations. RNA-seq analyses provided valuable insights into the molecular consequences of TTN variants, particularly in relation to splicing defects and nonsense-mediated RNA decay. In conclusion, this study reinforces the genotype-phenotype correlations between congenital myopathies and variants in TTN MTT-only exons, improves their molecular diagnosis, and provides a better understanding of their pathophysiology.

Alpha-tubulin 4A encoding gene (TUBA4A) has been associated with familial amyotrophic lateral sclerosis and frontotemporal dementia, based on identification of likely pathogenic variants in patients from distinct amyotrophic lateral sclerosis and frontotemporal dementia cohorts.By screening a multicentric French cohort of 448 unrelated probands presenting with cerebellar ataxia, we identified ultra-rare TUBA4A missense variants, all being absent from public databases and predicted pathogenic by multiple in silico tools. In addition, gene burden analyses in the 100 000 Genomes project (100KGP) showed enrichment of TUBA4A rare variants in the inherited ataxia group compared to controls [odds ratio: 57.0847 (10.2-576.7); P = 4.02 x10-7].Taken together, we report 12 patients presenting with spasticity and/or cerebellar ataxia and harbouring a predicted pathogenic TUBA4A missense mutation, including five confirmed de novo cases and a mutation previously reported in a large family presenting with spastic ataxia. Cultured fibroblasts from three patients harbouring distinct TUBA4A missense showed significant alterations in microtubule organization and dynamics, providing insight of TUBA4A variants pathogenicity.Our data confirm the identification of a hereditary spastic ataxia disease gene with variable age of onset, expanding the clinical spectrum of TUBA4A associated phenotypes. Benkirane et al. confirm the identification of a neurodegenerative disease associated with mutations in the gene TUBA4A, by describing 12 patients harbouring ultra-rare TUBA4A missense variants. Functional studies performed on skin biopsies from three patients revealed alterations in microtubule organization and dynamics.

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.

Purpose: Nonerythrocytic alpha II-spectrin (SPTAN1) variants have been previously associated with intellectual disability and epilepsy. We conducted this study to delineate the phenotypic spec-trum of SPTAN1 variants.Methods: We carried out SPTAN1 gene enrichment analysis in the rare disease component of the 100,000 Genomes Project and screened 100,000 Genomes Project, DECIPHER database, and GeneMatcher to identify individuals with SPTAN1 variants. Functional studies were performed on fibroblasts from 2 patients. Results: Statistically significant enrichment of rare (minor allele frequency < 1 x 10-5) probably damaging SPTAN1 variants was identified in families with hereditary ataxia (HA) or hereditary spastic paraplegia (HSP) (12/1142 cases vs 52/23,847 controls, p = 2.8 x 10-5). We identified 31 individuals carrying SPTAN1 heterozygous variants or deletions. A total of 10 patients presented with pure or complex HSP/HA. The remaining 21 patients had developmental delay and seizures. Irregular alpha II-spectrin aggregation was noted in fibroblasts derived from 2 patients with p.(Arg19Trp) and p.(Glu2207del) variants.Conclusion: We found that SPTAN1 is a genetic cause of neurodevelopmental disorder, which we classified into 3 distinct subgroups. The first comprises developmental epileptic encepha-lopathy. The second group exhibits milder phenotypes of developmental delay with or without seizures. The final group accounts for patients with pure or complex HSP/HA.(c) 2022 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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.

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. (c) 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.