Titin copy number variations associated with dominant inherited phenotypes.

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.

Deep Characterization of a Greek Patient with Desmin-Related Myofibrillar Myopathy and Cardiomyopathy.

Desmin is a class III intermediate filament protein highly expressed in cardiac, smooth and striated muscle. Autosomal dominant or recessive mutations in the desmin gene (DES) result in a variety of diseases, including cardiomyopathies and myofibrillar myopathy, collectively called desminopathies. Here we describe the clinical, histological and radiological features of a Greek patient with a myofibrillar myopathy and cardiomyopathy linked to the c.734A>G,p.(Glu245Gly) heterozygous variant in the DES gene. Moreover, through ribonucleic acid sequencing analysis in skeletal muscle we show that this variant provokes a defect in exon 3 splicing and thus should be considered clearly pathogenic.

Objective Evaluation of Clinical Actionability for Genes Involved in Myopathies: 63 Genes with a Medical Value for Patient Care.

The implementation of high-throughput diagnostic sequencing has led to the generation of large amounts of mutational data, making their interpretation more complex and responsible for long delays. It has been important to prioritize certain analyses, particularly those of "actionable" genes in diagnostic situations, involving specific treatment and/or management. In our project, we carried out an objective assessment of the clinical actionability of genes involved in myopathies, for which only few data obtained methodologically exist to date. Using the ClinGen Actionability criteria, we scored the clinical actionability of all 199 genes implicated in myopathies published by FILNEMUS for the "National French consensus on gene Lists for the diagnosis of myopathies using next generation sequencing". We objectified that 63 myopathy genes were actionable with the currently available data. Among the 36 myopathy genes with the highest actionability scores, only 8 had been scored to date by ClinGen. The data obtained through these methodological tools are an important resource for strategic choices in diagnostic approaches and the management of genetic myopathies. The clinical actionability of genes has to be considered as an evolving concept, in relation to progresses in disease knowledge and therapeutic approaches.

Development and Validation of a New Risk Prediction Score for Life-Threatening Ventricular Tachyarrhythmias in Laminopathies.

BACKGROUND: An accurate estimation of the risk of life-threatening (LT) ventricular tachyarrhythmia (VTA) in patients with LMNA mutations is crucial to select candidates for implantable cardioverter-defibrillator implantation. METHODS: We included 839 adult patients with LMNA mutations, including 660 from a French nationwide registry in the development sample, and 179 from other countries, referred to 5 tertiary centers for cardiomyopathies, in the validation sample. LTVTA was defined as (1) sudden cardiac death or (2) implantable cardioverter defibrillator-treated or hemodynamically unstable VTA. The prognostic model was derived using the Fine-Gray regression model. The net reclassification was compared with current clinical practice guidelines. The results are presented as means (SD) or medians [interquartile range]. RESULTS: We included 444 patients, 40.6 (14.1) years of age, in the derivation sample and 145 patients, 38.2 (15.0) years, in the validation sample, of whom 86 (19.3%) and 34 (23.4%) experienced LTVTA over 3.6 [1.0-7.2] and 5.1 [2.0-9.3] years of follow-up, respectively. Predictors of LTVTA in the derivation sample were: male sex, nonmissense LMNA mutation, first degree and higher atrioventricular block, nonsustained ventricular tachycardia, and left ventricular ejection fraction (https://lmna-risk-vta.fr). In the derivation sample, C-index (95% CI) of the model was 0.776 (0.711-0.842), and the calibration slope 0.827. In the external validation sample, the C-index was 0.800 (0.642-0.959), and the calibration slope was 1.082 (95% CI, 0.643-1.522). A 5-year estimated risk threshold ≥7% predicted 96.2% of LTVTA and net reclassified 28.8% of patients with LTVTA in comparison with the guidelines-based approach. CONCLUSIONS: In comparison with the current standard of care, this risk prediction model for LTVTA in laminopathies significantly facilitated the choice of candidates for implantable cardioverter defibrillators. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03058185.

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.

Phenotype variability and natural history of X-linked myopathy with excessive autophagy.

OBJECTIVE: X-linked myopathy with excessive autophagy (XMEA) linked to the VMA21 gene leads to autophagy failure with progressive vacuolation and atrophy of skeletal muscles. Current knowledge of this rare disease is limited. Our objective was to define the clinical, radiological, and natural history of XMEA. METHODS: We conducted a retrospective study collecting clinical, genetic, muscle imaging, and biopsy data of XMEA patients followed in France and reviewed the literature for additional cases. RESULTS: Eighteen males had genetically confirmed XMEA in France, carrying four different VMA21 variants. Mean age at disease onset was 9.4 ± 9.9 (range 1-40) years. In 14/18 patients (77.8%), onset occurred during childhood (< 15 years); however in four patients, the disease started in adulthood. Patients had anterior and medial compartment thigh muscle weakness, distal contractures (56.3%), elevated CK levels (1287.9 ± 757.8 U/l) and autophagic vacuoles with sarcolemmal features on muscle histopathology. Muscle MRI (n = 10) showed a characteristic pattern of lower limb muscle involvement. In 11 patients, outcome measures were available for an average follow-up period of 10.6 ± 9.8 years and six of them show disease progression. Mean change of functional outcomes was 0.5 ± 1.2 points for Brooke and 2.2 ± 2.5 points for Vignos score, 7/16 patients (43.8%) needed a walking aid and 3/16 (18.8%) were wheelchair-bound (median age of 40 years old, range 39-48). The variant c.164-7 T > G was associated with a later onset of symptoms. Respiratory insufficiency was common (57.1%) but cardiac involvement rare (12.5%). INTERPRETATION: XMEA has variable age of onset, but a characteristic clinical, histopathological, and muscle imaging presentation, guiding the diagnosis. Although slowly, motor disability progresses with time, and relevant genotype-phenotype correlations will help design future clinical trials.

LAMA2-Related Muscular Dystrophy: The Importance of Accurate Phenotyping and Brain Imaging in the Diagnosis of LGMD.

We report three siblings from a non-consanguineous family presenting with contractural limb-girdle phenotype with intrafamilial variability. Muscle MRI showed posterior thigh and quadriceps involvement with a sandwich-like sign. Whole-exome sequencing identified two compound heterozygous missense TTN variants and one heterozygous LAMA2 variant. Brain MRI performed because of concentration difficulties in one of the siblings evidenced white-matter abnormalities, subsequently found in the others. The genetic analysis was re-oriented, revealing a novel pathogenic intronic LAMA2 variant which confirmed the LAMA2-RD diagnosis. This work highlights the importance of a thorough clinical phenotyping and the importance of brain imaging, in order to orientate and interpret the genetic analysis.

A severe case of PLOD1-related kyphoscoliotic Ehlers-Danlos syndrome associated with several arterial and venous complications: A case report.

Kyphoscoliotic Ehlers-Danlos syndrome (kEDS) is a rare genetic disorder combining congenital hypotonia, congenital/early onset and progressive kyphoscoliosis, and generalized joint hypermobility. Vascular fragility is another characteristic of the disease rarely described. We report a severe case of kEDS-PLOD1 with several vascular complications leading to difficulties in disease management.

An early onset benign myopathy with glycogen storage caused by a de novo 1.4 Mb-deletion of chromosome 14.

Early onset myopathies are a clinically and histologically heterogeneous monogenic diseases linked to approximately 90 genes. Molecular diagnosis is challenging, especially in patients with a mild phenotype. We describe a 26-year-old man with neonatal hypotonia, motor delay and seizures during infancy, and non-progressive, mild muscular weakness in adulthood. Serum Creatine kinase level was normal. Whole-body muscle MRI showed thin muscles, and brain MRI was unremarkable. A deltoid muscle biopsy showed glycogen storage. WGS revealed a de novo 1.4 Mb-deletion of chromosome 14, confirmed by Array-CGH. This microdeletion causes the loss of ten genes including RALGAPA1, encoding for RalA, a regulator of glucose transporter 4 (GLUT4) expression at the membrane of myofibers. GLUT4 was overexpressed in patient's muscle. Here we highlight the importance to search for chromosomal alterations in the diagnostic workup of early onset myopathies.

Clinical and functional characterization of a long survivor congenital titinopathy patient with a novel metatranscript-only titin variant.

Congenital titinopathies are an emerging group of a potentially severe form of congenital myopathies caused by biallelic mutations in titin, encoding the largest existing human protein involved in the formation and stability of sarcomeres. In this study we describe a patient with a congenital myopathy characterized by multiple contractures, a rigid spine, non progressive muscular weakness, and a novel homozygous TTN pathogenic variant in a metatranscript-only exon: the c.36400A > T, p.Lys12134*. Muscle biopsies showed increased internalized nuclei, variability in fiber size, mild fibrosis, type 1 fiber predominance, and a slight increase in the number of satellite cells. RNA studies revealed the retention of intron 170 and 171 in the open reading frame, and immunoflourescence and western blot studies, a normal titin content. Single fiber functional studies showed a slight decrease in absolute maximal force and a cross-sectional area with no decreases in tension, suggesting that weakness is not sarcomere-based but due to hypotrophy. Passive properties of single fibers were not affected, but the observed increased calcium sensitivity of force generation might contribute to the contractural phenotype and rigid spine of the patient. Our findings provide evidence for a pathogenic, causative role of a metatranscript-only titin variant in a long survivor congenital titinopathy patient with distal arthrogryposis and rigid spine.

Novel comprehensive diagnostic strategy in Pitt-Hopkins syndrome: clinical score and further delineation of the TCF4 mutational spectrum.

Pitt-Hopkins syndrome (PTHS), characterized by severe intellectual disability and typical facial gestalt, is part of the clinical spectrum of Rett-like syndromes. TCF4, encoding a basic helix-loop-helix (bHLH) transcription factor, was identified as the disease-causing gene with de novo molecular defects. While PTHS appears to be a recognizable clinical entity, it seems to remain underdiagnosed, especially when facial gestalt is less typical. With the aim to facilitate the diagnosis of PTHS and to increase its rate and specificity, we have investigated 33 novel patients and defined a Clinical Diagnosis Score. Analysis of 112 individuals (79 previously reported and 33 novel patients) allowed us to delineate the TCF4 mutational spectrum, with 40% point mutations, 30% small deletions/insertions, and 30% deletions. Most of these were private mutations and generated premature stop codons. Missense mutations were localized in the bHLH domain, which is a mutational hotspot. No obvious difference was observed between patients harboring truncating, missense mutations, or deletions, further supporting TCF4 haploinsufficiency as the molecular mechanism underlying PTHS. In this study, we have summarized the current knowledge of TCF4 molecular pathology, reported all the mutations in the TCF4 database (http://www.LOVD.nl/TCF4), and present a novel and comprehensive diagnostic strategy for PTHS.

SEMA3A deletion in a family with Kallmann syndrome validates the role of semaphorin 3A in human puberty and olfactory system development.

BACKGROUND: Kallmann syndrome (KS) is a genetic disorder associating pubertal failure with congenitally absent or impaired sense of smell. KS is related to defective neuronal development affecting both the migration of olfactory nerve endings and GnRH neurons. The discovery of several genetic mutations responsible for KS led to the identification of signaling pathways involved in these processes, but the mutations so far identified account for only 30% of cases of KS. Here, we attempted to identify new genes responsible for KS by using a pan-genomic approach. METHODS: From a cohort of 120 KS patients, we selected 48 propositi with no mutations in known KS genes. They were analyzed by comparative genomic hybridization array, using Agilent 105K oligonucleotide chips with a mean resolution of 50 kb. RESULTS: One propositus was found to have a heterozygous deletion of 213 kb at locus 7q21.11, confirmed by real-time qPCR, deleting 11 of the 17 SEMA3A exons. This deletion cosegregated in the propositus' family with the KS phenotype, that was transmitted in autosomal dominant fashion and was not associated with other neurological or non-neurological clinical disorders. SEMA3A codes for semaphorin 3A, a protein that interacts with neuropilins. Mice lacking semaphorin 3A expression have been showed to have a Kallmann-like phenotype. CONCLUSIONS: SEMA3A is therefore a new gene whose loss-of-function is involved in KS. These findings validate the specific role of semaphorin 3A in the development of the olfactory system and in neuronal control of puberty in humans.

Molecular characterization of 1q44 microdeletion in 11 patients reveals three candidate genes for intellectual disability and seizures.

Patients with a submicroscopic deletion at 1q43q44 present with intellectual disability (ID), microcephaly, craniofacial anomalies, seizures, limb anomalies, and corpus callosum abnormalities. However, the precise relationship between most of deleted genes and the clinical features in these patients still remains unclear. We studied 11 unrelated patients with 1q44 microdeletion. We showed that the deletions occurred de novo in all patients for whom both parents' DNA was available (10/11). All patients presented with moderate to severe ID, seizures and non-specific craniofacial anomalies. By oligoarray-based comparative genomic hybridization (aCGH) covering the 1q44 region at a high resolution, we obtained a critical deleted region containing two coding genes-HNRNPU and FAM36A-and one non-coding gene-NCRNA00201. All three genes were expressed in different normal human tissues, including in human brain, with highest expression levels in the cerebellum. Mutational screening of the HNRNPU and FAM36A genes in 191 patients with unexplained isolated ID did not reveal any deleterious mutations while the NCRNA00201 non-coding gene was not analyzed. Nine of the 11 patients did not present with microcephaly or corpus callosum abnormalities and carried a small deletion containing HNRNPU, FAM36A, and NCRNA00201 but not AKT3 and ZNF238, two centromeric genes. These results suggest that HNRNPU, FAM36A, and NCRNA00201 are not major genes for microcephaly and corpus callosum abnormalities but are good candidates for ID and seizures.

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.

Diagnostic interest of whole-body MRI in early- and late-onset LAMA2 muscular dystrophies: a large international cohort.

BACKGROUND: LAMA2-related muscular dystrophy (LAMA2-RD) encompasses a group of recessive muscular dystrophies caused by mutations in the LAMA2 gene, which codes for the alpha-2 chain of laminin-211 (merosin). Diagnosis is straightforward in the classic congenital presentation with no ambulation and complete merosin deficiency in muscle biopsy, but is far more difficult in milder ambulant individuals with partial merosin deficiency. OBJECTIVE: To investigate the diagnostic utility of muscle imaging in LAMA2-RD using whole-body magnetic resonance imaging (WBMRI). RESULTS: 27 patients (2-62 years, 21-80% with acquisition of walking ability and 6 never ambulant) were included in an international collaborative study. All carried two pathogenic mutations, mostly private missense changes. An intronic variant (c.909 + 7A > G) was identified in all the Chilean cases. Three patients (two ambulant) showed intellectual disability, epilepsy, and brain structural abnormalities. WBMRI T1w sequences or T2 fat-saturated images (Dixon) revealed abnormal muscle fat replacement predominantly in subscapularis, lumbar paraspinals, gluteus minimus and medius, posterior thigh (adductor magnus, biceps femoris, hamstrings) and soleus. This involvement pattern was consistent for both ambulant and non-ambulant patients. The degree of replacement was predominantly correlated to the disease duration, rather than to the onset or the clinical severity. A "COL6-like sandwich sign" was observed in several muscles in ambulant adults, but different involvement of subscapularis, gluteus minimus, and medius changes allowed distinguishing LAMA2-RD from collagenopathies. The thigh muscles seem to be the best ones to assess disease progression. CONCLUSION: WBMRI in LAMA2-RD shows a homogeneous pattern of brain and muscle imaging, representing a supportive diagnostic tool.

Two new cases of interstitial 7q35q36.1 deletion including CNTNAP2 and KMT2C.

BACKGROUND: Terminal deletions of the long arm of chromosome 7 are well known and frequently associated with syndromic holoprosencephaly due to the involvement of the SHH (aliases HHG1, SMMCI, TPT, TPTPS, and MCOPCB5) gene region. However, interstitial deletions including CNTNAP2 (aliases Caspr2, KIAA0868, and NRXN4) and excluding the SHH region are less common. METHODS: We report the clinical and molecular characterization associated with pure 7q35 and 7q35q36.1 deletion in two unrelated patients as detected by oligonucleotide-based array-CGH analysis. RESULTS: The common clinical features were abnormal maternal serum screening during first-trimester pregnancy, low occipitofrontal circumference at birth, hypotonia, abnormal feet, developmental delay, impaired language development, generalized seizures, hyperactive behavior, friendly personality, and cranio-facial dysmorphism. Both deletions occurred de novo and sequencing of CNTNAP2, a candidate gene for epilepsy and autism showed absence of mutation on the contralateral allele. CONCLUSION: Combined haploinsufficiency of GALNTL5 (alias GalNAc-T5L), CUL1, SSPO (aliases SCO-spondin, KIAA0543, and FLJ36112), AOC1 (alias DAO), RHEB, and especially KMT2C (alias KIAA1506 and HALR) with monoallelic disruption of CNTNAP2 may explain neurologic abnormalities, hypotonia, and exostoses. Haploinsufficiency of PRKAG2 (aliases AAKG, AAKG2, H91620p, WPWS, and CMH6) and KCNH2 (aliases Kv11.1, HERG, and erg1) genes may be responsible of long QT syndrome observed for one patient.

A novel COL1A1 variant in a family with clinical features of hypermobile Ehlers-Danlos syndrome that proved to be a COL1-related overlap disorder.

COL1-related overlap disorder is a condition, which is not yet considered as part of the 2017 EDS classification. However, it should be investigated as an alternative diagnosis for any patient with hypermobile EDS. This could allow providing appropriate genetic counseling.

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.