Comparison between a novel helical and a posterior ankle-foot orthosis on gait in people with unilateral foot drop: a randomised crossover trial.

BACKGROUND: Neuromuscular disease and peripheral neuropathy may cause drop foot with or without evertor weakness. We developed a helical-shaped, non-articulated ankle-foot orthosis (AFO) to provide medial-lateral stability while allowing mobility, to improve gait capacity. Our aim was to evaluate the effect of the helical AFO (hAFO) on functional gait capacity (6-min walk test) in people with peripheral neuropathy or neuromuscular disease (NMD) causing unilateral drop foot and compare with a posterior leaf spring AFO (plsAFO). Secondary aims were to compare functional mobility, 3D kinematic and kinetic gait variables and satisfaction between the AFOs. METHODS: Single centre, randomised crossover trial from January to July 2017 in 20 individuals (14 with peripheral neuropathy and 6 with NMD, 12 females, mean age 55.6 years, SD 15.3); 10 wore the hAFO for the first week and 10 wore the plsAFO before switching for the second week. The 6-min walk test (6MWT), Timed Up and Go (TUG) test and 3D gait analysis were evaluated with the hAFO, the plsAFO and shoes only (noAFO) at inclusion and 1 week after wearing each orthosis. Satisfaction was evaluated with the Quebec user evaluation of satisfaction with assistive technology (QUEST). RESULTS: Median [interquartile range] 6MWT distance was greater with the hAFO (444 m [79]) than the plsAFO (389 m [135], P < 0.001, Hedge's g = 0.6) and noAFO (337 m [91], P < 0.001, g = 0.88). TUG time was shorter with the hAFO (8.1 s [2.8]) than the plsAFO (9.5 s [2.6], P < 0.001, g = - 0.5) and noAFO (10.0 s [2.6]), P < 0.001, g = - 0.6). The plsAFO limited plantarflexion during the loading response (plsAFO - 7.5 deg [6.0] vs. noAFO -13.0 deg [10.0], P = 0.0007, g = - 1.0) but the hAFO did not (- 11.0 deg [5.1] vs. noAFO, P = 0.05, g = - 0.5). Quasi-stiffness was lower for the hAFO than plsAFO (P = 0.009, g = - 0.7). The dimensionless eversion moment was higher (though not significantly) with the hAFO than noAFO. Neither orthosis reduced ankle power (P = 0.34). Median total QUEST score was higher for the hAFO (4.7 [0.7]) than the plsAFO (3.6 [0.8]) (P < 0.001, g = 1.9). CONCLUSIONS: The helical orthosis significantly and considerably improved functional gait performance, did not limit ankle mobility, increased lateral stability, though not significantly, and was associated with greater patient satisfaction than the posterior leaf spring orthosis. Trial registration The trial began before registration was mandatory.

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.

Clinical features of homozygous FIG4-p.Ile41Thr Charcot-Marie-Tooth 4J patients.

We describe the clinical, electrodiagnostic, and genetic findings of three homozygous FIG4-c.122T>C patients suffering from Charcot-Marie-Tooth disease type 4J (AR-CMT-FIG4). This syndrome usually involves compound heterozygosity associating FIG4-c.122T>C, a hypomorphic allele coding an unstable FIG4-p.Ile41Thr protein, and a null allele. While the compound heterozygous patients presenting with early onset usually show rapid progression, the homozygous patients described here show the signs of relative clinical stability. As FIG4 activity is known to be dose dependent, these patients' observations could suggest that the therapeutic perspective of increasing levels of the protein to improve the phenotype of AR-CMT-FIG4-patients might be efficient.

Hearing loss in inherited peripheral neuropathies: Molecular diagnosis by NGS in a French series.

BACKGROUND: The most common inherited peripheral neuropathy is Charcot-Marie-Tooth disease (CMT), with a prevalence of 1/2500. Other symptoms can be associated to the condition, such as hearing loss. Currently, no global hearing impairment assessment has been determined, and the physiopathology is not well known. METHODS: The aim of the study was to analyze among a French series of 3,412 patients with inherited peripheral neuropathy (IPN), the ones who also suffer from hearing loss, to establish phenotype-genotype correlations. An NGS strategy for IPN one side and nonsyndromic hearing loss (NSHL) on the other side, were performed. RESULTS: Hearing loss (HL) was present in only 44 patients (1.30%). The clinical data of 27 patients were usable. Demyelinating neuropathy was diagnosed in 15 cases and axonal neuropathy in 12 cases. HL varied from mild to profound. Five cases of auditory neuropathy were noticed. Diagnosis was made for 60% of these patients. Seven novel pathogenic variants were discovered in five different genes: PRPS1; MPZ; SH3TC2; NEFL; and ABHD12. Two patients with PMP22 variant, had also an additional variant in COCH and MYH14 respectively. No pathogenic variant was found at the DFNB1 locus. Genotype-phenotype correlations do exist, especially with SH3TC2, PRPS1, ABHD12, NEFL, and TRPV4. CONCLUSION: Involvement of PMP22 is not enough to explain hearing loss in patients suffering from IPN. HL can be due to cochlear impairment and/or auditory nerve dysfunction. HL is certainly underdiagnosed, and should be evaluated in every patient suffering from IPN.

New PRPS1 variant p.(Met68Leu) located in the dimerization area identified in a French CMTX5 patient.

BACKGROUND: CMTX5 is characterized by peripheral neuropathy, early-onset sensorineural hearing impairment, and optic neuropathy. Only seven variants have been reported and no genotype-phenotype correlations have yet been established. PRPS1 has a crystallographic structure, as it is composed of three dimers that constitute a hexamer. METHODS: Next-generation sequencing (NGS) was performed using a custom 92-gene panel designed for the diagnosis of Charcot-Marie-Tooth (CMT) and associated neuropathies. RESULTS: We report the case of a 35-year-old male, who had presented CMT and hearing loss since childhood associated to bilateral optic neuropathy without any sign of retinitis pigmentosa. A new hemizygous variant on chromosomic position X:106,882,604, in the PRPS1 gene, c.202A > T, p.(Met68Leu) was found. This change is predicted to lead to an altered affinity between the different subunits in the dimer, thereby may prevent the hexamer formation. CONCLUSION: CMTX5 is probably under-diagnosed, as an overlap among the different features due to PRPS1 exists. Patients who developed polyneuropathy associated to sensorineural deafness and optic atrophy during childhood should be assessed for PRPS1.

A Reliable Targeted Next-Generation Sequencing Strategy for Diagnosis of Myopathies and Muscular Dystrophies, Especially for the Giant Titin and Nebulin Genes.

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.

Gender as a Modifying Factor Influencing Myotonic Dystrophy Type 1 Phenotype Severity and Mortality: A Nationwide Multiple Databases Cross-Sectional Observational Study.

BACKGROUND: Myotonic Dystrophy type 1 (DM1) is one of the most heterogeneous hereditary disease in terms of age of onset, clinical manifestations, and severity, challenging both medical management and clinical trials. The CTG expansion size is the main factor determining the age of onset although no factor can finely predict phenotype and prognosis. Differences between males and females have not been specifically reported. Our aim is to study gender impact on DM1 phenotype and severity. METHODS: We first performed cross-sectional analysis of main multiorgan clinical parameters in 1409 adult DM1 patients (>18 y) from the DM-Scope nationwide registry and observed different patterns in males and females. Then, we assessed gender impact on social and economic domains using the AFM-Téléthon DM1 survey (n = 970), and morbidity and mortality using the French National Health Service Database (n = 3301). RESULTS: Men more frequently had (1) severe muscular disability with marked myotonia, muscle weakness, cardiac, and respiratory involvement; (2) developmental abnormalities with facial dysmorphism and cognitive impairment inferred from low educational levels and work in specialized environments; and (3) lonely life. Alternatively, women more frequently had cataracts, dysphagia, digestive tract dysfunction, incontinence, thyroid disorder and obesity. Most differences were out of proportion to those observed in the general population. Compared to women, males were more affected in their social and economic life. In addition, they were more frequently hospitalized for cardiac problems, and had a higher mortality rate. CONCLUSION: Gender is a previously unrecognized factor influencing DM1 clinical profile and severity of the disease, with worse socio-economic consequences of the disease and higher morbidity and mortality in males. Gender should be considered in the design of both stratified medical management and clinical trials.

Chloride channel dysfunction study in myotonic dystrophy type 1 using repeated short exercise tests.

INTRODUCTION: This exploratory study aimed to evaluate the electrophysiological profiles of patients with myotonic dystrophy type 1 (DM1) and to assess their correlations with genotype and phenotype. METHODS: Twenty-two patients with genetically confirmed DM1 were included. Global motor testing score, severity of myotonia, occurrence of cardiac disturbances, and CTG repeat number were recorded. All patients underwent repeated short exercise tests after 7 min of cooling. RESULTS: Two trajectories could be distinguished following 3 periods of exercise, although most clearly following the third exercise period. Cardiac disturbances were more common among patients who had a B-type trajectory (larger decrement in compound muscle potential amplitude and slower recovery) following the third exercise period. CONCLUSIONS: While the electrophysiological pattern in each profile appeared to confirm chloride muscle channel impairment, the B-type trajectory may suggest dysfunction of other muscle channels in DM1 and their link with cardiac disturbances. Muscle Nerve 54: 104-109, 2016.

Low penetrance in facioscapulohumeral muscular dystrophy type 1 with large pathological D4Z4 alleles: a cross-sectional multicenter study.

BACKGROUND: Facioscapulohumeral muscular dystrophy type 1(FSHD1) is an autosomal dominant disorder associated with the contraction of D4Z4 less than 11 repeat units (RUs) on chromosome 4q35. Penetrance in the range of the largest alleles is poorly known. Our objective was to study the penetrance of FSHD1 in patients carrying alleles ranging between 6 to10 RUs and to evaluate the influence of sex, age, and several environmental factors on clinical expression of the disease. METHODS: A cross-sectional multicenter study was conducted in six French and one Swiss neuromuscular centers. 65 FSHD1 affected patients carrying a 4qA allele of 6-10 RUs were identified as index cases (IC) and their 119 at-risk relatives were included. The age of onset was recorded for IC only. Medical history, neurological examination and manual muscle testing were performed for each subject. Genetic testing determined the allele size (number of RUs) and the 4qA/4qB allelic variant. The clinical status of relatives was established blindly to their genetic testing results. The main outcome was the penetrance defined as the ratio between the number of clinically affected carriers and the total number of carriers. RESULTS: Among the relatives, 59 carried the D4Z4 contraction. At the clinical level, 34 relatives carriers were clinically affected and 25 unaffected. Therefore, the calculated penetrance was 57% in the range of 6-10 RUs. Penetrance was estimated at 62% in the range of 6-8 RUs, and at 47% in the range of 9-10 RUs. Moreover, penetrance was lower in women than men. There was no effect of drugs, anesthesia, surgery or traumatisms on the penetrance. CONCLUSIONS: Penetrance of FSHD1 is low for largest alleles in the range of 9-10 RUs, and lower in women than men. This is of crucial importance for genetic counseling and clinical management of patients and families.

Detection of TRIM32 deletions in LGMD patients analyzed by a combined strategy of CGH array and massively parallel sequencing.

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.

Prevalence of type 1 Brugada ECG pattern after administration of Class 1C drugs in patients with type 1 myotonic dystrophy: Myotonic dystrophy as a part of the Brugada syndrome.

BACKGROUND: Both type 1 myotonic dystrophy (MD1) and Brugada syndrome (BrS) may be complicated by conduction disturbances and sudden death. Spontaneous BrS has been observed in MD1 patients, but the prevalence of drug-induced BrS in MD1 is unknown. OBJECTIVE: The purpose of this study was to prospectively assess the prevalence of type 1 ST elevation as elicited during pharmacologic challenge with Class 1C drugs in a subgroup of MD1 patients and to further establish correlations with ECG and electrophysiologic variables and prognosis. METHODS: From a group of unselected 270 MD1 patients, ajmaline or flecainide drug challenge was performed in a subgroup of 44 patients (27 men, median age 43 years) with minor depolarization/repolarization abnormalities suggestive of possible BrS. The presence of type 1 ST elevation after drug challenge was correlated to clinical, ECG, and electrophysiologic variables. RESULTS: Eight of 44 patients (18%) presented with BrS after drug challenge. BrS was seen more often in men (26% vs 6%, P = .09) and was related to younger age (35 vs 48 years, P = .07). BrS was not correlated to symptoms, baseline ECG, HV interval, results of signal-averaged ECG, or abnormalities on ambulatory recordings. MD1 patients with BrS had longer corrected QT intervals, greater increase in PR interval after drug challenge, and higher rate of inducible ventricular arrhythmias (62% vs 21%, P = .03). Twelve patients were implanted with a pacemaker and 5 with an implantable cardioverter-defibrillator. Significant bradycardia did not occur in any patients, and malignant ventricular arrhythmia never occurred during median 7-year follow-up (except 1 hypokalemia-related ventricular fibrillation). CONCLUSION: BrS is elicited by a Class 1 drug in 18% of MD1 patients presenting with minor depolarization/repolarization abnormalities at baseline, but the finding seems to be devoid of a prognostic role.

Recessive TTN truncating mutations define novel forms of core myopathy with heart disease.

Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery-Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.

Genotype-phenotype correlations in Charcot-Marie-Tooth disease type 2 caused by mitofusin 2 mutations.

BACKGROUND: Mutations in the gene encoding mitofusin 2 (MFN2) cause Charcot-Marie-Tooth disease type 2 (CMT2), with heterogeneity concerning severity and associated clinical features. OBJECTIVE: To describe MFN2 mutations and associated phenotypes in patients with hereditary motor and sensory neuropathy (HMSN). DESIGN: Direct sequencing of the MFN2 gene and clinical investigations of patients with MFN2 mutations. SETTING: Molecular genetics laboratory of a university hospital and the Limoges National Referral Center for Rare Peripheral Neuropathies. PATIENTS: One hundred fifty index patients with HMSN and a median motor nerve conduction velocity of 25 m/s or greater and without mutations in the genes encoding connexin 32 and myelin protein zero. MAIN OUTCOME MEASURES: Results of genetic analyses and phenotypic observations. RESULTS: Twenty different missense mutations were identified in 20 index patients. Mutation frequency was 19 of 107 (17.8%) in patients with CMT2 and 1 of 43 (2.3%) in patients with a median motor nerve conduction velocity less than 38 m/s. Four patients had proven de novo mutations, 8 families had autosomal dominant inheritance, and 3 had autosomal recessive inheritance. The remaining 5 patients were sporadic cases with heterozygous mutations. Phenotypes varied from mild forms to early-onset severe forms. Additional features were encountered in 8 patients (32%). Six patients underwent sural nerve biopsy: electronic microscopy showed prominent mitochondrial abnormalities on longitudinal sections. CONCLUSIONS: MFN2 mutations are a frequent cause of CMT2, with variable severity and either dominant or recessive inheritance. MFN2 gene testing must be a first-line analysis in axonal HMSN irrespective of the mode of inheritance or the severity of the peripheral neuropathy.

Striking phenotypic variability in two familial cases of myosin storage myopathy with a MYH7 Leu1793pro mutation.

Myosin Storage Myopathies (MSM) have emerged as a new group of inherited myopathies with heterogenous clinical severity and age of onset. We have identified in a woman and her daughter, a pLeu1793Pro mutation in MYH7. This mutation has already been reported to be associated with MSM presenting as neonatal hypotony. Our index case complained of proximal muscle weakness at age 30. Her daughter presented at birth with a cardiomyopathy without any skeletal muscle involvement. This report underlines the clinical variability of MSM even with a given mutation or in a same family.

Danon's disease (X-linked vacuolar cardiomyopathy and myopathy): a case with a novel Lamp-2 gene mutation.

Herein, we report a new case of Danon's disease in a 41-year-old Frenchman. This patient displays the typical clinical triad, with cardiomyopathy, mental retardation and myopathy, and a vacuolar myopathy without acid alpha-glucosidase deficiency. He has also developed a diffuse chorio-capillary ocular atrophy, and represents the second case of successful heart transplantation in this lysosomal disease. Interestingly, analysis of LAMP-2 protein expression in cultured fibroblasts revealed a primary deficiency of this lysosomal membrane protein. This defect resulted from a yet undescribed deletion in exon 7 of lamp-2 gene.