Rapid Molecular Diagnosis of Genetically Inherited Neuromuscular Disorders Using Next-Generation Sequencing Technologies.

Neuromuscular diseases are genetically highly heterogeneous, and differential diagnosis can be challenging. Over a 3-year period, we prospectively analyzed 268 pediatric and adult patients with a suspected diagnosis of inherited neuromuscular disorder (INMD) using comprehensive gene-panel analysis and next-generation sequencing. The rate of diagnosis increased exponentially with the addition of genes to successive versions of the INMD panel, from 31% for the first iteration (278 genes) to 40% for the last (324 genes). The global mean diagnostic rate was 36% (97/268 patients), with a diagnostic turnaround time of 4-6 weeks. Most diagnoses corresponded to muscular dystrophies/myopathies (68.37%) and peripheral nerve diseases (22.45%). The most common causative genes, TTN, RYR1, and ANO5, accounted for almost 30% of the diagnosed cases. Finally, we evaluated the utility of the differential diagnosis tool Phenomizer, which established a correlation between the phenotype and molecular findings in 21% of the diagnosed patients. In summary, comprehensive gene-panel analysis of all genes implicated in neuromuscular diseases facilitates a rapid diagnosis and provides a high diagnostic yield.

LAMA2 gene mutation update: Toward a more comprehensive picture of the laminin-α2 variome and its related phenotypes.

Congenital muscular dystrophy type 1A (MDC1A) is one of the main subtypes of early-onset muscle disease, caused by disease-associated variants in the laminin-α2 (LAMA2) gene. MDC1A usually presents as a severe neonatal hypotonia and failure to thrive. Muscle weakness compromises normal motor development, leading to the inability to sit unsupported or to walk independently. The phenotype associated with LAMA2 defects has been expanded to include milder and atypical cases, being now collectively known as LAMA2-related muscular dystrophies (LAMA2-MD). Through an international multicenter collaborative effort, 61 new LAMA2 disease-associated variants were identified in 86 patients, representing the largest number of patients and new disease-causing variants in a single report. The collaborative variant collection was supported by the LOVD-powered LAMA2 gene variant database (https://www.LOVD.nl/LAMA2), updated as part of this work. As of December 2017, the database contains 486 unique LAMA2 variants (309 disease-associated), obtained from direct submissions and literature reports. Database content was systematically reviewed and further insights concerning LAMA2-MD are presented. We focus on the impact of missense changes, especially the c.2461A > C (p.Thr821Pro) variant and its association with late-onset LAMA2-MD. Finally, we report diagnostically challenging cases, highlighting the relevance of modern genetic analysis in the characterization of clinically heterogeneous muscle diseases.

Screening for Pompe disease in a Portuguese high risk population.

Pompe disease is a rare metabolic disorder with available enzymatic replacement therapy. Contrasting with the classic infantile form, the others subtypes have a heterogeneous presentation that makes an early and accurate diagnosis difficult. We conducted a prospective, multicenter, observational study to identify undiagnosed patients. During a one-year period, patients followed in Portuguese neuromuscular outpatient clinics with proximal muscle weakness affecting upper and/or lower limbs, hyperCKemia in two or more determinations or hypotonia and hyperCKemia, were screened for acid α-glucosidase deficiency by dried blood spots. Lysosomal acid-alpha-1,4-glucosidase activity was determined by tandem mass spectrometry and positive results were confirmed by molecular study. From the 99 patients screened, Pompe disease was confirmed in 4, with age of onset ranging from 2.5 to 48 years, all with limb girdle muscle weakness, corresponding to a frequency of 4% in our cohort and 4.9% of limb girdle muscle weakness. Screening for Pompe disease in high risk populations, using dried blood spots, was already performed in some European populations. Apart from two negative Scandinavian studies, positive cases were confirmed in 2.8-7.9% of patients presenting with limb girdle muscle weakness and in 0-2.5% with isolated hyperCKemia.

Type 1 diabetes and GAD65 limbic encephalitis: a case report of a 10-year-old girl.

Limbic encephalitis is a rare neurological disorder that may be difficult to recognize. Clinical features include memory impairment, temporal lobe seizures and affective disturbance. We report the case of a 10-year-old girl with type 1 diabetes mellitus that presented with seizures, depressed mood and memory changes. The diagnosis of glutamic acid decarboxylase 65 (GAD65) mediated limbic encephalitis relied on cerebral magnetic resonance imaging lesions and high serological and cerebrospinal fluid GAD65-antibodies titers. High-dose steroidal therapy was started with clinical improvement. Relapse led to a second high-dose steroid treatment followed by rituximab with remission. A correlation between serum GAD65-antibodies levels and symptoms was found, demonstrating GAD65-antibodies titers may be useful for clinical follow-up and immunotherapy guidance. This report raises awareness of this serious neurological condition that may be associated with type 1 diabetes, underlining the importance of an early diagnosis and prompt treatment for a better prognosis.

New splicing mutation in the choline kinase beta (CHKB) gene causing a muscular dystrophy detected by whole-exome sequencing.

Muscular dystrophies (MDs) are a group of hereditary muscle disorders that include two particularly heterogeneous subgroups: limb-girdle MD and congenital MD, linked to 52 different genes (seven common to both subgroups). Massive parallel sequencing technology may avoid the usual stepwise gene-by-gene analysis. We report the whole-exome sequencing (WES) analysis of a patient with childhood-onset progressive MD, also presenting mental retardation and dilated cardiomyopathy. Conventional sequencing had excluded eight candidate genes. WES of the trio (patient and parents) was performed using the ion proton sequencing system. Data analysis resorted to filtering steps using the GEMINI software revealed a novel silent variant in the choline kinase beta (CHKB) gene. Inspection of sequence alignments ultimately identified the causal variant (CHKB:c.1031+3G>C). This splice site mutation was confirmed using Sanger sequencing and its effect was further evaluated with gene expression analysis. On reassessment of the muscle biopsy, typical abnormal mitochondrial oxidative changes were observed. Mutations in CHKB have been shown to cause phosphatidylcholine deficiency in myofibers, causing a rare form of CMD (only 21 patients reported). Notwithstanding interpretative difficulties that need to be overcome before the integration of WES in the diagnostic workflow, this work corroborates its utility in solving cases from highly heterogeneous groups of diseases, in which conventional diagnostic approaches fail to provide a definitive diagnosis.

New variants, challenges and pitfalls in DMD genotyping: implications in diagnosis, prognosis and therapy.

Molecular characterization of patients with Duchenne or Becker muscular dystrophies is essential for establishing a differential diagnosis, allowing appropriate clinical follow-up, patient management and genetic counseling. In light of the recent mutation-based therapeutic approaches, DMD gene analysis has gained further relevance. Owing to the size and complexity of the DMD gene and the diversity of mutation types, molecular analysis is not always a straightforward task requiring the combination of several methodologies. Our national genetic diagnostic service genetically characterized 308 dystrophinopathy patients (284 unrelated families), leading to the identification of 175 distinct mutations, including 39 unpublished variants. These studies revealed several potential diagnostic pitfalls (because of technical limitations or related with DMD's genetic heterogeneity) that may be overlooked even considering the international disease-specific diagnostic guidelines. Comprehensive analysis involved expression studies at the mRNA level, the identification of splicing changes and ultimately providing evidence for apparent exceptions to the reading-frame rule. Besides increasing the mutation detection rate, this detailed molecular characterization is indispensable for the identification of suitable candidates for the new mutation-centered therapies. As patient registries are internationally recognized as essential for clinical trial recruitment, this led us to develop the Portuguese Duchenne and Becker Muscular Dystrophy registry in collaboration with the Translational Research in Europe-Assessment and Treatment of Neuromuscular Diseases network.

A 15-year follow-up of first unprovoked seizures: a prospective study of 200 children.

Epilepsy is associated with an extended spectrum of behaviour, psychiatric problems, and learning difficulties. The aim of this study was to establish the natural history of children with first unprovoked seizures. We studied prospectively 200 children under the age of 11 years who attended hospital emergency with a first unprovoked seizure. Demographic variables, personal and family history, neurological examination, EEG, psychiatric, and cognitive and educational profiles were analysed. Patients who developed epilepsy were characterised with respect to: time to relapse, remission rate, duration of epilepsy, neuroimaging, aetiology, epileptic syndrome, and therapeutic regimen. These results were compared to data of patients who had a single seizure over a follow-up period of 15 years. Thirty percent of children who had a first unprovoked seizure developed epilepsy. Partial seizure type was a statistically significant variable for the development of epilepsy. An EEG with epileptic abnormalities proved to be the main risk factor for recurrence. Fifteen years later, the group with epilepsy exhibited a 2.6 greater risk of psychiatric and academic comorbidities, compared to the group without epilepsy.

Reviewing Large LAMA2 Deletions and Duplications in Congenital Muscular Dystrophy Patients.

BACKGROUND: Congenital muscular dystrophy (CMD) type 1A (MDC1A) is caused by recessive mutations in laminin-α2 (LAMA2) gene. Laminin-211, a heterotrimeric glycoprotein that contains the α2 chain, is crucial for muscle stability establishing a bond between the sarcolemma and the extracellular matrix. More than 215 mutations are listed in the locus specific database (LSDB) for LAMA2 gene (May 2014). OBJECTIVE: A limited number of large deletions/duplications have been reported in LAMA2. Our main objective was the identification of additional large rearrangements in LAMA2 found in CMD patients and a systematic review of cases in the literature and LSDB. METHODS: In four of the fifty-two patients studied over the last 10 years, only one heterozygous mutation was identified, after sequencing and screening for a frequent LAMA2 deletion. Initial screening of large mutations was performed by multiplex ligation-dependent probe application (MLPA). Further characterization implied several techniques: long-range PCR, cDNA and Southern-blot analysis. RESULTS: Three novel large deletions in LAMA2 and the first pathogenic large duplication were successfully identified, allowing a definitive molecular diagnosis, carrier screening and prenatal diagnosis. A total of fifteen deletions and two duplications previously reported were also reviewed. Two possible mutational "hotspots" for deletions may exist, the first encompassing exons 3 and 4 and second in the 3' region (exons 56 to 65) of LAMA2. CONCLUSIONS: Our findings show that this type of mutation is fairly frequent (18.4% of mutated alleles) and is underestimated in the literature. It is important to include the screening of large deletions/duplications as part of the genetic diagnosis strategy.

Autosomal recessive axonal neuropathy with neuromyotonia: a rare entity.

BACKGROUND: Autosomal recessive axonal neuropathy with neuromyotonia is a recently described entity associated to the HINT1 gene, encoding histidine triad nucleotide-binding protein 1. PATIENT: The authors report a Portuguese 16-year-old girl of Roma ethnicity, descendant of consanguineous parents, with progressive distal muscular atrophy and weakness, beginning at age 6. After several years of extensive investigation with inconclusive results, clinical myotonia was identified. Electrophysiologic studies revealed neuromyotonia associated with a severe chronic predominantly motor axonal neuropathy and homozygous mutation (c.334 C > A, p.H112 N) in HINT1 was detected. CONCLUSION: This report emphasizes the late onset of clinical myotonia essential to the diagnosis.

Pyridoxine-dependent epilepsy due to antiquitin deficiency: achieving a favourable outcome.

We report 4 pyridoxine-dependent epilepsy patients in which good outcome was determined in three. The 4 patients were male and aged from 7 to 24 years old (from three unrelated Caucasian families). A clinical diagnosis of neonatal pyridoxine-dependent epilepsy was confirmed by biochemical and genetic studies. Clinical evaluation was performed and medical records were reviewed for therapy implementation and management, neurodevelopment outcome, magnetic resonance imaging, and electroencephalography. All were taking pyridoxine treatment and were seizure-free. Elevated urinary alpha-aminoadipic semialdehyde excretion was found in all patients. Antiquitin gene analysis identified a large homozygous deletion in one patient and two heterozygous mutations in the others. Treatment with pyridoxine should be attempted for all cases of infantile and childhood refractory epilepsy, as has been the case over the last 20 years. Currently, urinary alpha-aminoadipic semialdehyde is a reliable biomarker of pyridoxine-dependent epilepsy, even under pyridoxine treatment. Detection of mutations in the antiquitin gene, encoding alpha-aminoadipic semialdehyde dehydrogenase, establishes the diagnosis and allows for adequate genetic counselling.

[Cerebral creatine deficiency syndromes]. / Síndromes de deficiência cerebral de creatina.

INTRODUCTION: Creatine deficiency syndromes are a recently described group of diseases characterized by inborn errors of creatine metabolism. Clinical features include a spectrum of neurodevelopment disorders of diverse severity. They are characterized by low levels of cerebral creatine caused by different pathogenic mutations concerning the genes coding for creatine synthesis enzymes [arginine: glicyne amidinotransferase (AGAT, EC 2.1.4.1) and guanidinoacetate methyltansferase (GAMT, EC 2.1.1.2)], AGAT and GAMT, respectively, or its transporter (CT1 deficiency), SLC6A8. Enzymatic deficiencies are transmitted as autosomal recessive traits, whereas the transporter deficit is X-linked. OBJECTIVES: To characterize the clinical and laboratorial presentation, diagnosis and treatment of cerebral creatine deficiency patients, followed in Hospital Pediátrico Carmona da Mota. The awareness of these inborn errors of metabolism as neurological disorders, namely of neurodevelopment, among the medical community is a secondary aim of the present work. METHODS AND MATERIAL: Retrospective analysis of the clinical files of patients followed in our Hospital and diagnosed with cerebral creatine deficiency syndrome. RESULTS: Twelve patients belonging to seven different families were diagnosed with creatine deficiency syndromes. Five presented GAMT deficiency and seven CT1 deficiency. Present ages are 2 to 38 years old. The most common clinical presentations were: global development delay in seven patients (two with epilepsy), and speech delay in two patients. Only one patient had communication and social interaction dysfunction. In all, global development delay in the range of intellectual delay was identified. The pathognomonic pattern of cerebral creatine deficiency in the brain image was demonstrated in eight patients. Pathogenic mutations in GAMT or SLC6A8 genes were identified in all cases. CONCLUSIONS: The suspicion of cerebral creatine depletion must be considered in all children presenting unexplained global psychomotor development delay. Pre-symptomatic therapy has shown promising results, especially in GAMT deficiency patients. The high rate of asymptomatic carriers of GAMT mutations in our population makes this disorder eligible to neonatal screening in Portugal.

[Neuropsychological evaluation in children with epilepsy: attention and executive functions in temporal lobe epilepsy]. / Evaluación neuropsicológica en niños con epilepsia: atención y funciones ejecutivas en epilepsia del lóbulo temporal.

INTRODUCTION: Attention and executive functions correspond to important areas of cognitive functioning associated with the frontal cortex. The study of attention and executive functions in children with epilepsy has focused on characterizing the group with frontal lobe epilepsy. Still, recent studies have identified deficits in these areas also in children with temporal lobe epilepsy (TLE). AIM: To investigate attention and executive functions in a group of children with TLE, also considering the influence of clinical variables (age at onset of epilepsy and evolution of seizures). SUBJECTS AND METHODS: Attention and executive functions were studied in a group of 24 children with TLE, aged 7-15 years and compared with 24 control children of the same age and socio-cultural level. Subjects were assigned the following tasks: Cancellation Task, Trail Making Test, Tower of London and Phonemic Verbal Fluency. RESULTS: The group with TLE performed significantly lower on selective, sustained and divided attention and on phonemic verbal fluency. In markers concerning commissions and omissions there were no differences between groups in any of the performed tests. A significant slowing of processing speed was reported. In addition, patients with earlier age at onset of epilepsy had more difficulties in sustained attention and planning abilities. CONCLUSION: These results sustain the need for evaluating and monitoring the area of attention, executive functions and processing speed in children and adolescents with temporal lobe epilepsy, above all those with earlier age at onset of epilepsy.

Value of brain magnetic resonance imaging in mitochondrial respiratory chain disorders.

Mitochondrial respiratory chain (MRC) disorders have variable clinical manifestations which are mainly neurologic. Diagnosis in children is more complex than in adults because the classic phenotype, ragged red fibers, and mtDNA mutations are rarely seen in children. Moreover, clinical manifestations of disease in developing brains are less explicit. Although not specific, neuroimaging may be contributory to the diagnosis of these disorders in pediatric patients. Brain magnetic resonance images were reviewed for 133 pediatric patients investigated for a MRC disorder at a single center over a period of 10 years (1997-2006), in an attempt to identify distinctive neuroimaging features of MRC defects. Patients fit into four groups, according to the Bernier criteria: definite (63 cases), probable (53 cases), possible (7 cases) and unlikely diagnosis (10 cases). Brain atrophy (41 cases), supratentorial white matter lesions (14 cases), basal ganglia involvement (9 cases), and delayed myelination (9 cases) were the most frequent anomalies in the definite group, and 8 patients presented Leigh syndrome. Neuroimaging findings of the 63 children in the definite group were compared with the remainder and with those in the possible and unlikely groups. There were no significant differences in brain images between the groups analyzed, and therefore no distinctive brain imaging features were identified specific for MRC disorders.

Novel synonymous substitution in POMGNT1 promotes exon skipping in a patient with congenital muscular dystrophy.

Walker-Warburg syndrome, muscle-eye-brain disease, Fukuyama congenital muscular dystrophy, congenital muscular dystrophy type 1C, and congenital muscular dystrophy type 1D are overlapping clinical entities belonging to a subgroup of the congenital muscular dystrophies (CMD), collectively designated dystroglycanopathies, in which the common underlying defect is hypoglycosylation of alfa-dystroglycan. Currently, six different genes are known to be implicated in these diseases: POMT1, POMT2, POMGNT1, FCMD, FKRP, and LARGE. We report the molecular characterization of a patient presenting clinical features of CMD and reduced immunostaining for alfa-dystroglycan in muscle. Three candidate genes (FCMD, POMT1 and POMGNT1) were analyzed, and a total of 18 sequence variants were detected: 15 polymorphisms in POMT1 [including three unreported single nucleotide polymorphisms (SNPs)], two polymorphisms in FCMD, and the exonic silent mutation c.636C > T in POMGNT1. Expression analysis revealed that this apparently silent mutation compromises correct premessenger RNA (mRNA) splicing, promoting skipping of the entire exon 7, with a consequent frameshift. In silico analysis of this mutation did not predict alterations in the canonical splice sequences, but rather the creation of a new exonic splice silencer. The recognition of such disease-causing elements may contribute to the further understanding of RNA processing and assist mutation screening in routine diagnosis, where such changes may be underestimated. To aid clinical diagnosis, we generated publicly available LOVD-powered Locus Specific Databases for these three genes and recorded all known sequence variants ( http://www.dmd.nl ).

Spinal muscular atrophy--noninvasive ventilatory support in pediatrics.

The deterioration of the respiratory function in children suffering from degenerative neuromuscular disease is the main cause of the high mortality rate associated with these diseases. Noninvasive ventilation (NIV) has reduced the morbidity and mortality due to respiratory insufficiency in these children. However, the use of support ventilation in some cases of spinal muscular atrophy (SMA) is still controversial. A retrospective study of 22 patients suffering from SMA who were followed up in the Paediatric Hospital of Coimbra is presented: 7 of type I, 11 of type II, and 4 of type III. In 17 of these cases, non-invasive ventilation by mask was begun, and in 3 of them NIV was applied for prophylactic purposes. The 7 children with SMA type I began NIV when they were 13 months of age on average (3 months-3 years); 5 of them died, between 1 and 15 months after the beginning of the ventilation. Of the 11 children with SMA type II, 8 were submitted to NIV and one died 22 months later. Three of the children in this group began NIV in a prophylactic way, and in all of them a decrease in the thoracic deformity was observed. Of the 4 patients of type III, 2 of them were submitted to non-invasive ventilation. In all of the symptomatic cases, a decrease in the frequency and severity of respiratory infections was observed, after ventilation was started. The respiratory support with NIV may improve the quality of life of children suffering from SMA as well as prolong their life expectancies. In SMA type I, whose clinical manifestations are precocious and whose prognostic is very serious, the application of this support has been debated.