Alternative splicing expands the clinical spectrum of NDUFS6-related mitochondrial disorders.

PURPOSE: We describe 3 families with Charcot-Marie-Tooth neuropathy (CMT), harboring a homozygous NDUFS6 NM_004553.6:c.309+5G>A variant previously linked to fatal Leigh syndrome. We aimed to characterize clinically and molecularly the newly identified patients and understand the mechanism underlying their milder phenotype. METHODS: The patients underwent extensive clinical examinations. Exome sequencing was done in 4 affected individuals. The functional effect of the c.309+5G>A variant was investigated in patient-derived EBV-transformed lymphoblasts at the complementary DNA, protein, and mitochondrial level. Alternative splicing was evaluated using complementary DNA long-read sequencing. RESULTS: All patients presented with early-onset, slowly progressive axonal CMT, and nystagmus; some exhibited additional central nervous system symptoms. The c.309+5G>A substitution caused the expression of aberrantly spliced transcripts and negligible levels of the canonical transcript. Immunoblotting showed reduced levels of mutant isoforms. No detectable defects in mitochondrial complex stability or bioenergetics were found. CONCLUSION: We expand the clinical spectrum of NDUFS6-related mitochondrial disorders to include axonal CMT, emphasizing the clinical and pathophysiologic overlap between these 2 clinical entities. This work demonstrates the critical role that alternative splicing may play in modulating the severity of a genetic disorder, emphasizing the need for careful consideration when interpreting splice variants and their implications on disease prognosis.

Clinical characteristics and outcomes of thymoma-associated myasthenia gravis.

BACKGROUND AND PURPOSE: Prognosis of myasthenia gravis (MG) in patients with thymoma is not well established. Moreover, it is not clear whether thymoma recurrence or unresectable lesions entail a worse prognosis of MG. METHODS: This multicenter study was based on data from a Spanish neurologist-driven MG registry. All patients were aged >18 years at onset and had anti-acetylcholine receptor antibodies. We compared the clinical data of thymomatous and nonthymomatous patients. Prognosis of patients with recurrent or nonresectable thymomas was assessed. RESULTS: We included 964 patients from 15 hospitals; 148 (15.4%) had thymoma-associated MG. Median follow-up time was 4.6 years. At onset, thymoma-associated MG patients were younger (52.0 vs. 60.4 years, p < 0.001), had more generalized symptoms (odds ratio [OR]: 3.02, 95% confidence interval [CI]: 1.95-4.68, p < 0.001) and more severe clinical forms according to the Myasthenia Gravis Foundation of America (MGFA) scale (OR: 1.6, 95% CI: 1.15-2.21, p = 0.005). Disease severity based on MGFA postintervention status (MGFA-PIS) was higher in thymomatous patients at 1 year, 5 years, and the end of follow-up. Treatment refractoriness and mortality were also higher (OR: 2.28, 95% CI: 1.43-3.63, p = 0.001; hazard ratio: 2.46, 95% CI: 1.47-4.14, p = 0.001). Myasthenic symptoms worsened in 13 of 27 patients with recurrences, but differences in long-term severity were not significant. Fifteen thymomatous patients had nonresectable thymomas with worse MGFA-PIS and higher mortality at the end of follow-up. CONCLUSIONS: Thymoma-associated MG patients had more severe myasthenic symptoms and worse prognosis. Thymoma recurrence was frequently associated with transient worsening of MG, but long-term prognosis did not differ from nonrecurrent thymoma. Patients with nonresectable thymoma tended to present severe forms of MG.

Ultrasonography of cervical nerve roots: cross-sectional reference values according to age.

INTRODUCTION: The aim of this study is to describe the normal cross-sectional area (CSA) and appearance of cervical nerve roots in ultrasound, correlating it to age and other patient somatic parameters. METHODS: One hundred healthy volunteers were included. We aimed to achieve uniform representation throughout all age groups. Ultrasound of the cervical nerve roots was performed bilaterally. CSA and margins description were obtained. RESULTS: C5 nerve, 8.32 ± 2.30; C6 nerve, 11.88 ± 3.36; C7 nerve, 12.79 ± 3.85; C8 nerve, 11.20 ± 3.45. Significant correlation between CSA and age was demonstrated, but not for body mass index. Blurred margins were present in up to 23.71% cervical nerves, more frequently in older individuals and in C7 nerve. DISCUSSION: If ultrasound morphology of cervical nerve roots is used as a diagnostic parameter, the normal range of CSA values and percentage of blurred margins according to age should be considered.

Muscle MRI in a large cohort of patients with oculopharyngeal muscular dystrophy.

BACKGROUND AND OBJECTIVE: Oculopharyngeal muscular dystrophy (OPMD) is a genetic disorder caused by an abnormal expansion of GCN triplets within the PABPN1 gene. Previous descriptions have focused on lower limb muscles in small cohorts of patients with OPMD, but larger imaging studies have not been performed. Previous imaging studies have been too small to be able to correlate imaging findings to genetic and clinical data. METHODS: We present cross-sectional, T1-weighted muscle MRI and CT-scan data from 168 patients with genetically confirmed OPMD. We have analysed the pattern of muscle involvement in the disease using hierarchical analysis and presented it as heatmaps. Results of the scans were correlated with genetic and clinical data. RESULTS: Fatty replacement was identified in 96.7% of all symptomatic patients. The tongue, the adductor magnus and the soleus were the most commonly affected muscles. Muscle pathology on MRI correlated positively with disease duration and functional impairment. CONCLUSIONS: We have described a pattern that can be considered characteristic of OPMD. An early combination of fat replacement in the tongue, adductor magnus and soleus can be helpful for differential diagnosis. The findings suggest the natural history of the disease from a radiological point of view. The information generated by this study is of high diagnostic value and important for clinical trial development.

A unicenter, prospective study of Guillain-Barré syndrome in Spain.

OBJECTIVE: We report a prospective study analysing clinical characteristics, subtyping and prognosis in Guillain-Barré syndrome (GBS). METHOD: The study was based on consecutive GBS patients admitted between 2009 and 2017. Disability was serially assessed using the GBS disability scale. RESULTS: Fifty-six GBS patients were identified with an average age of 55 years (range, 5-86 years) and a male/female ratio of 2.1. The interval to nadir was <7 days in 59% of cases, and 7 to 28 days in the remainder; at nadir, 35.5% of patients were able to walk unaided, and 64.5% did not. Mechanical ventilation was needed in 20% of cases. There were two fatal cases. Clinical variants included paraparetic GBS seven cases, Miller Fisher syndrome one case, and acute sensory ataxic neuropathy (ASAN) one case. Serial electrophysiology showed a demyelinating pattern in 62.5% of cases, axonal in 28.5%, inexcitable in 1.8%, equivocal in 1.8%, and normal in 5.4%. Very early (1 to 4 days after onset) electrophysiology was done in 18 patients; equivocal or normal features in six of them evolved into an axonal pattern in four. Reversible conduction failure of sensitive nerves occurred in ASAN. Antiganglioside antibodies were only detected in axonal GBS. At 24-month follow-up, functional outcome did not differ between demyelinating and axonal GBS. Clinico-pathological correlation in an early fatal case is reported. CONCLUSIONS: This GBS study demonstrates comparable clinical features to previous investigations from well-defined populations. There was a relatively high prevalence of axonal GBS. We provide new pathophysiological insights on nerve conduction alterations.

Characterising the phenotype and mode of inheritance of patients with inherited peripheral neuropathies carrying MME mutations.

BACKGROUND: Mutations in the metalloendopeptidase (MME) gene were initially identified as a cause of autosomal recessive Charcot-Marie-Tooth disease type 2 (CMT2). Subsequently, variants in MME were linked to other late-onset autosomal dominant polyneuropathies. Thus, our goal was to define the phenotype and mode of inheritance of patients carrying changes in MME. METHODS: We screened 197 index cases with a hereditary neuropathy of the CMT type or distal hereditary motor neuropathy (dHMN) and 10 probands with familial amyotrophic lateral sclerosis (fALS) using a custom panel of 119 genes. In addition to the index case subjects, we also studied other clinically and/or genetically affected and unaffected family members. RESULTS: We found 17 variants in MME in a total of 20 index cases, with biallelic MME mutations detected in 13 cases from nine families (three in homozygosis and six in compound heterozygosis) and heterozygous variants found in 11 families. All patients with biallelic variants had a similar phenotype, consistent with late-onset axonal neuropathy. Conversely, the phenotype of patients carrying heterozygous mutations was highly variable [CMT type 1 (CMT1), CMT2, dHMN and fALS] and mutations did not segregate with the disease. CONCLUSION: MME mutations that segregate in an autosomal recessive pattern are associated with a late-onset CMT2 phenotype, yet we could not demonstrate that MME variants in heterozygosis cause neuropathy. Our data highlight the importance of establishing an accurate genetic diagnosis in patients carrying MME mutations, especially with a view to genetic counselling.

Charcot-Marie-Tooth disease type 2G redefined by a novel mutation in LRSAM1.

OBJECTIVE: To identify the unknown genetic cause in a large pedigree previously classified with a distinct form of axonal Charcot-Marie-Tooth disease type 2G (CMT2G) and to explore its transcriptional consequences. METHODS: Clinical reevaluation of the pedigree was performed, followed by linkage analysis with the redefined disease statuses, and whole genome and exome sequencing. The impact of the mutation was investigated by immunoblotting and transcriptome sequencing. RESULTS: Thirteen affected individuals over 3 generations displayed mild and quiescent lower-limb axonal sensorimotor neuropathy. Magnetic resonance imaging (MRI) of lower-limb musculature systematically showed fatty atrophy in clinical and subclinical mutation carriers. We redefined the disease-linked region to chr9q31.3-q34.2 and subsequently identified a novel missense variant in the E3 ubiquitin-protein ligase LRSAM1 (p.Cys694Tyr). Unlike previous reports, we demonstrated in patients' lymphoblasts that the mutation does not influence overall protein levels of LRSAM1, nor of its ubiquitylation target TSG101. The mutation is associated with several transcriptional changes, including a significant upregulation of another E3 ubiquitin-protein ligase, NEDD4L, and of TNFRSF21, a key regulator of axonal degeneration. INTERPRETATION: Our findings demonstrate that the isolated genetic entity CMT2G is caused by a missense mutation in LRSAM1 and should be reclassified as CMT2P. MRI of lower-limb musculature can be used to detect minimal signs of the disease. Transcriptome analysis of patients' cells highlights novel molecular players associated with LRSAM1 dysfunction, and reveals pathways and therapeutic targets shared with amyotrophic lateral sclerosis and Alzheimer disease. Ann Neurol 2016;80:823-833.

Peripheral neuropathy in complex inherited diseases: an approach to diagnosis.

Peripheral neuropathy is a common finding in patients with complex inherited neurological diseases and may be subclinical or a major component of the phenotype. This review aims to provide a clinical approach to the diagnosis of this complex group of patients by addressing key questions including the predominant neurological syndrome associated with the neuropathy, for example, spasticity, the type of neuropathy and the other neurological and non-neurological features of the syndrome. Priority is given to the diagnosis of treatable conditions. Using this approach, we associated neuropathy with one of three major syndromic categories: (1) ataxia, (2) spasticity and (3) global neurodevelopmental impairment. Syndromes that do not fall easily into one of these three categories can be grouped according to the predominant system involved in addition to the neuropathy, for example, cardiomyopathy and neuropathy. We also include a separate category of complex inherited relapsing neuropathy syndromes, some of which may mimic Guillain-Barré syndrome, as many will have a metabolic aetiology and be potentially treatable.

Biomarkers predict outcome in Charcot-Marie-Tooth disease 1A.

BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited neuropathy, a debilitating disease without known cure. Among patients with CMT1A, disease manifestation, progression and severity are strikingly variable, which poses major challenges for the development of new therapies. Hence, there is a strong need for sensitive outcome measures such as disease and progression biomarkers, which would add powerful tools to monitor therapeutic effects in CMT1A. METHODS: We established a pan-European and American consortium comprising nine clinical centres including 311 patients with CMT1A in total. From all patients, the CMT neuropathy score and secondary outcome measures were obtained and a skin biopsy collected. In order to assess and validate disease severity and progression biomarkers, we performed qPCR on a set of 16 animal model-derived potential biomarkers in skin biopsy mRNA extracts. RESULTS: In 266 patients with CMT1A, a cluster of eight cutaneous transcripts differentiates disease severity with a sensitivity and specificity of 90% and 76.1%, respectively. In an additional cohort of 45 patients with CMT1A, from whom a second skin biopsy was taken after 2-3 years, the cutaneous mRNA expression of GSTT2, CTSA, PPARG, CDA, ENPP1 and NRG1-Iis changing over time and correlates with disease progression. CONCLUSIONS: In summary, we provide evidence that cutaneous transcripts in patients with CMT1A serve as disease severity and progression biomarkers and, if implemented into clinical trials, they could markedly accelerate the development of a therapy for CMT1A.

Electromyographic tendon reflex recording: An accurate and comfortable method for diagnosis of charcot-marie-tooth disease type 1a.

INTRODUCTION: We analyzed the utility of tendon reflex (T-reflex) testing in Charcot-Marie-Tooth disease type 1A (CMT1A). METHODS: A total of 82 subjects from 27 unrelated CMT1A pedigrees were evaluated prospectively. The series also comprised 28 adult healthy controls. Electrophysiology included evaluation of biceps T-reflex and soleus T-reflex. RESULTS: Seventy-one individuals (62 adults and 9 children) had clinical and electrophysiological features of CMT1A. The remaining 11 (8 adults and 3 children) were unaffected. On electrophysiological testing, the biceps T-reflex was elicited in 58 of 62 (93%) adult CMT1A patients and in all 9 affected children. Latencies of the biceps T-reflex were always markedly prolonged, and a cut-off limit of 16.25 ms clearly separated adult patients and controls or unaffected kin adult individuals. In affected children, the soleus T-reflex latency was also prolonged when compared with age and height normative data. CONCLUSION: T-reflex testing is an accurate diagnostic technique for CMT1A patients.

MFN2 deletion of exons 7 and 8: founder mutation in the UK population.

Mitofusin 2 (MFN2) mutations are the most common cause of axonal Charcot-Marie-Tooth disease (CMT2). The majority are inherited in an autosomal dominant manner but recessive and semi-dominant kindreds have also been described. We previously reported a deletion of exons 7 and 8 resulting in nonsense-mediated decay, segregating with disease when present in trans with another pathogenic MFN2 mutation. Detailed clinical and electrophysiological data on a series of five affected patients from four kindreds and, when available, their parents and relatives were collected. MFN2 Sanger sequencing, multiplex ligation probe amplification, and haplotype analysis were performed. A severe early-onset CMT phenotype was seen in all cases: progressive distal weakness, wasting, and sensory loss from infancy or early childhood. Optic atrophy (four of five) and wheelchair dependency in childhood were common (four of five). All were compound heterozygous for a deletion of exons 7 and 8 in MFN2 with another previously reported pathogenic mutation (Phe216Ser, Thr362Met, and Arg707Trp). Carrier parents and relatives were unaffected (age range: 24-82 years). Haplotype analysis confirmed that the deletion had a common founder in all families.

Expanding the Clinical Spectrum of DRP2-Associated Charcot-Marie-Tooth Disease.

BACKGROUND AND OBJECTIVES: Germline truncating variants in the DRP2 gene (encoding dystrophin-related protein 2) cause the disruption of the periaxin-DRP2-dystroglycan complex and have been linked to Charcot-Marie-Tooth disease. However, the causality and the underlying phenotype of the genetic alterations are not clearly defined. METHODS: This cross-sectional retrospective observational study includes 9 patients with Charcot-Marie-Tooth disease (CMT) with DRP2 germline variants evaluated at 6 centers throughout Spain. RESULTS: We identified 7 Spanish families with 4 different DRP2 likely pathogenic germline variants. In agreement with an X-linked inheritance, men harboring hemizygous DRP2 variants presented with an intermediate form of CMT, whereas heterozygous women were asymptomatic. Symptom onset was variable (36.6 ± 16 years), with lower limb weakness and multimodal sensory loss producing a mild-to-moderate functional impairment. Nerve echography revealed an increase in the cross-sectional area of nerve roots and proximal nerves. Lower limb muscle magnetic resonance imaging confirmed the presence of a length-dependent fatty infiltration. Immunostaining in intradermal nerve fibers demonstrated the absence of DRP2 and electron microscopy revealed abnormal myelin thickness that was also detectable in the sural nerve sections. DISCUSSION: Our findings support the causality of DRP2 pathogenic germline variants in CMT and further define the phenotype as a late-onset sensory and motor length-dependent neuropathy, with intermediate velocities and thickening of proximal nerve segments.

Epidemiological and genetic features of anti-3­hydroxy-3-methylglutaryl-CoA reductase necrotizing myopathy: Single-center experience and literature review.

OBJECTIVE: To characterize the demographic, genetic, clinical, and serological features of patients with anti-3­hydroxy-3-methylglutaryl-CoA reductase (HMGCR) immune-mediated necrotizing myopathy (IMNM) in a region of northern Spain. METHODS: Study of all patients diagnosed with anti-HMGCR IMNM during a 5-year period at a reference hospital in northern Spain. Besides clinical and laboratory data, we analyzed the genetic influence of HLA genes and the rs4149056 (c.521T>C) single nucleotide polymorphism (SNP) in the SLCO1B1 gene. RESULTS: 8 patients (5 women, 3 men) with a mean ± SD age of 64.9 ± 7.3 years, fulfilled the criteria for anti-HMGCR IMNM. The incidence rate was 0.6 per 100.000 person-years and the prevalence 3 per 100.000 population. All patients had been exposed to statins. All of them had predominant lower limb proximal and symmetric muscle weakness that was severe in 2 and had elevated serum CK levels with a median [IQR] of 4488 [2538-9194] IU/L. Serum 25­hydroxy vitamin D levels were decreased in all patients in whom it was determined. The 3 patients with a previous diagnosis of hypothyroidism had abnormal levels of TSH at the time of diagnosis. All patients experienced improvement with different schemes of immunosuppressive therapy. Noteworthy, 7 of 8 patients carried the HLA-DRB1*11 allele. The frequency of the rs4149056 C allele in the SLCO1B1 gene (12.5%) was similar to that of the general population. CONCLUSION: In northern Spain, anti-HMGCR IMNM preferentially affects people over 50 years of age who are carriers of the HLA-DRB1*11 allele and take statins. Both low vitamin D levels and hypothyroidism may play a potential predisposing role in the development of this disease.

Drug-refractory myasthenia gravis: Clinical characteristics, treatments, and outcome.

OBJECTIVE: To describe the clinical characteristics and outcomes in patients with refractory myasthenia gravis (MG) and to determine the effectiveness and side effects of the drugs used for their treatment. METHODS: This observational retrospective cross-sectional multicenter study was based on data from the Spanish MG Registry (NMD-ES). Patients were considered refractory when their MG Foundation of America post-interventional status (MGFA-PIS) was unchanged or worse after corticosteroids and two or more other immunosuppressive agents. Clinical and immunologic characteristics of drug-refractory patients, efficiency and toxicity of drugs used, and outcome (MGFA-PIS) at end of follow-up were studied. RESULTS: We included 990 patients from 15 hospitals. Eighty-four patients (68 of 842 anti-acetylcholine receptor [AChR], 5 of 26 anti-muscle-specific tyrosine kinase [MusK], 10 of 120 seronegative, and 1 of 2 double-seropositive patients) were drug refractory. Drug-refractory patients were more frequently women (p < 0.0001), younger at onset (p < 0.0001), and anti-MuSK positive (p = 0.037). Moreover, they more frequently presented a generalized form of the disease, bulbar symptoms, and life-threatening events (p < 0.0001; p = 0.018; and p = 0.002, respectively) than non-drug-refractory patients. Mean follow-up was 9.8 years (SD 4.5). Twenty-four (50%) refractory patients had side effects to one or more of the drugs. At the end of follow-up, 42.9% of drug-refractory patients (42.6% of anti-AChR, 100% of anti-MuSK, and 10% of seronegative patients) and 79.8% of non-drug-refractory patients (p < 0.0001) achieved remission or had minimal manifestations. Eighty percent of drug-refractory-seronegative patients did not respond to any drug tested. INTERPRETATION: In this study, 8.5% of MG patients were drug-refractory. New more specific drugs are needed to treat drug-refractory MG patients.

Overview of treatments used in transthyretin-related hereditary amyloidosis: a systematic review.

OBJECTIVE: To carry out a systematic review of the literature to analyse the efficacy and safety of treatments available or under investigation for amyloidosis due to mutations in the transthyretin gene (ATTR). METHODS: A bibliographic search was carried out in the following electronic databases up to September 2017: PubMed, Cochrane Library and EMBASE. The inclusion criteria were: efficacy and/or safety studies conducted in humans, studies that included treatments, including treatments in the research phase, and studies that included 10 or more patients. RESULTS: A total of 21 articles were included; 16 were clinical trials, eight of them (50%) phase III trials, and five were observational studies. Of the total number of studies selected, 11 were on tafamidis, four on diflunisal, two on liver transplantation, two on patisiran and two on other therapeutic alternatives. Of the 11 studies related to the drug, the pivotal trial, the results of its two extension studies and an additional post hoc analysis were selected. In addition, two phase III trials were included in specific populations, two phase II studies, one safety study and two observational studies. Regarding the four included studies related to the drug, one was the pivotal trial that gave the indication to diflunisal, another a safety summary of the pivotal trial, and the other two trials were carried out in specific populations, one in a Japanese population and another phase I trial in cardiac amyloidosis in the USA. As far as other alternatives are concerned, of the six studies included in this section, two were related to liver transplantation, two to patisiran and two to different therapeutic alternatives. CONCLUSIONS: Sufficient evidence has not been found that demonstrates superiority among the available oral alternatives, diflunisal or tafamidis, in the treatment of ATTR. Direct comparisons between both drugs and pharmacoeconomic studies would be necessary to select the most efficient treatment.

Very early Guillain-Barré syndrome: A clinical-electrophysiological and ultrasonographic study.

Objectives: Using recent optimized electrodiagnostic criteria sets, we primarily aimed at verifying the accuracy of the initial electrophysiological test in very early Guillain-Barré syndrome (VEGBS), ≤4 days of onset, compared with the results of serial electrophysiology. Our secondary objective was to correlate early electrophysiological results with sonographic nerve changes. Methods: This is a retrospective study based on consecutive VEGBS patients admitted to the hospital. Each patient had serial nerve conduction studies (NCS) in at least 4 nerves. Initial NCS were done within 4 days after onset, and serial ones from the second week onwards. Electrophysiological recordings of each case were re-evaluated, GBS subtype being established accordingly. Nerve ultrasonography was almost always performed within 2 weeks after onset. Results: Fifteen adult VEGBS patients were identified with a mean age of 57.8 years. At first NCS, VEGBS sub-typing was only possible in 3 (20%) cases that showed an axonal pattern, the remaining patterns being mixed (combining axonal and demyelinating features) in 6 (40%), equivocal in 5 (33.3%), and normal in 1 (6.7%). Upon serial NCS, 7 (46.7%) cases were categorized as acute demyelinating polyneuropathy, 7 (46.7%) as axonal GBS, and 1 (6.6%) as unclassified syndrome. Antiganglioside reactivity was detected in 5 out of the 7 axonal cases. Nerve US showed that lesions mainly involved the ventral rami of scanned cervical nerves. Conclusions: Serial electrophysiological evaluation is necessary for accurate VEGBS subtype classification. Ultrasonography helps delineate the topography of nerve changes. Significance: We provide new VEGBS pathophysiological insights into nerve conduction alterations within the first 4 days of the clinical course.