Metabolic Myopathies: Experience of a Reference Center of Inherited Metabolic Diseases.

INTRODUCTION: Metabolic myopathies (MM) are a heterogeneous group of genetic disorders affecting metabolic pathways involved in energy production during rest, exercise and physiologic stress (fever, fasting, …). Impairments in the pathways of glycolysis/ glycogenolysis, fatty acid transport/oxidation or in the mitochondrial respiratory chain present primarily with exercise intolerance, myalgias, weakness, cramps, or rhabdomyolysis. Depending on aetiology, the diagnosis can be made through neonatal screening, pre-symptomatic or in the set of clinical manifestations for which a high level of suspicion is important. METHODS: Retrospective descriptive study of the clinical, biochemical, and molecular features of patients with a confirmed diagnosis of MM followed by the multidisciplinary team of the Reference Center of Inherited Metabolic Diseases of Centro Hospitalar Universitário de Lisboa Central from 2009 to 2022. RESULTS: Twenty-three patients with MM were included: 9 (39%) glycogen storage diseases (7 McArdle and 2 Pompe), 7 (30%) fatty acid oxidation disorders (3 CPT2, 3 LCHAD and 1 MAD deficiencies), 6 (26%) mitochondrial disease with significant muscle involvement (2 Pearson, 1 Kearns Sayre, 1 VARS2, 1 SUCLA2 and 1 MT-TL1 deficiencies), and 1 myoadenylate deaminase deficiency. Ages varied from 15 months to 35 years. Eighteen (78%) patients were diagnosed by clinical symptoms, 3 by newborn screening (LCHAD) and 2 were asymptomatic (1 Pompe and 1 McArdle). Frequent symptoms were rhabdomyolysis triggered by illness or exercise 12 (52%), fatigue 11 (48%), exercise intolerance 10 (43%), and myalgia 9 (43%). Eight (35%) patients (LCHAD and mitochondrial) had multisystemic involvement. In 20 (87%) patients, the diagnosis was confirmed by biochemical and/or genetic analysis and 3 (McArdle) by muscle biopsy. CONCLUSION: MM are a heterogeneous set of disorders, but a careful history may guide the differential diagnosis among biochemical pathways and other etiologies. Nowadays, molecular testing has become a powerful tool for diagnosis confirmation, surpassing muscular biopsy in most cases. Accurate diagnosis is important to identify who may benefit from specific therapeutic options, such as enzyme replacement therapy, restricted diets, emergency regime and cofactors. All patients benefit from adequate lifestyle modifications, individualized exercise prescription, nutritional intervention, and genetic counselling.

A Novel Genetic Variant in MBD5 Associated with Severe Epilepsy and Intellectual Disability: Potential Implications on Neural Primary Cilia.

Disruptions in the MBD5 gene have been linked with an array of clinical features such as global developmental delay, intellectual disability, autistic-like symptoms, and seizures, through unclear mechanisms. MBD5 haploinsufficiency has been associated with the disruption of primary cilium-related processes during early cortical development, and this has been reported in many neurodevelopmental disorders. In this study, we describe the clinical history of a 12-year-old child harboring a novel MBD5 rare variant and presenting psychomotor delay and seizures. To investigate the impact of MBD5 haploinsufficiency on neural primary cilia, we established a novel patient-derived cell line and used CRISPR-Cas9 technology to create an isogenic control. The patient-derived neural progenitor cells revealed a decrease in the length of primary cilia and in the total number of ciliated cells. This study paves the way to understanding the impact of MBD5 haploinsufficiency in brain development through its potential impact on neural primary cilia.

Adding evidence to the role of NEUROG1 in congenital cranial dysinnervation disorders.

Congenital cranial dysinnervation disorders (CCDDs) are a heterogeneous group of neurodevelopmental phenotypes caused by a primary disturbance of innervation due to deficient, absent, or misguided cranial nerves. Although some CCDDs genes are known, several clinical phenotypes and their aetiologies remain to be elucidated. We describe a 12-year-old boy with hypotonia, developmental delay, sensorineural hearing loss, and keratoconjunctivitis due to lack of corneal reflex. He had a long expressionless face, severe oromotor dysfunction, bilateral agenesis/severe hypoplasia of the VIII nerve with marked atresia of the internal auditory canals and cochlear labyrinth malformation. Trio-exome sequencing identified a homozygous loss of function variant in the NEUROG1 gene (NM_006161.2: c.202G > T, p.Glu68*). NEUROG1 is considered a causal candidate for CCDDs based on (i) the previous report of a patient with a homozygous gene deletion and developmental delay, deafness due to absent bilateral VIII nerves, and severe oromotor dysfunction; (ii) a second patient with a homozygous NEUROG1 missense variant and corneal opacity, absent corneal reflex and intellectual disability; and (iii) the knockout mouse model phenotype which highly resembles the disorder observed in humans. Our findings support the growing compelling evidence that loss of NEUROG1 leads to a very distinctive disorder of cranial nerves development.

Leigh syndrome with atypical cerebellar lesions.

Leigh Syndrome is a neurodegenerative disorder caused by mitochondrial dysfunction, with significant phenotypic and genetic heterogeneity. It usually presents in early life, with a severe prognosis. It can be caused by more than 75 different gene mutations, of nuclear and mitochondrial origin, involving all respiratory chain complexes, with less than 25% of Leigh syndrome having mitochondrial DNA mutations. The typical pathologic hallmarks are focal, bilateral, and symmetric lesions in the basal ganglia, thalamus, cerebellum, cerebral white matter and spinal cord gray matter, usually with T2WI and FLAIR hyperintensity. The basal ganglia and thalami frequently present with a pattern of cytotoxic edema. We present one case with clinical and analytical features consistent with Leigh Syndrome, with peculiar imaging features, showing dominant cerebellar edematous changes with unexpected petechial component suggestive of microangiopathy. To our knowledge, these features are unreported and suggest the existence of microvascular lesions. Based on the reported imaging findings, we propose that Leigh Syndrome should be added to the differential diagnosis of acute cerebellitis.

Borrelia lusitaniae Infection Mimicking Headache, Neurologic Deficits, and Cerebrospinal Fluid Lymphocytosis.

Headache with neurologic deficits and cerebrospinal fluid lymphocytosis (HaNDL) is a rare headache syndrome included in the Classification of Headache of the International Headache Society as a "headache attributed to non-infectious inflammatory intracranial disease." We report one 15-year-old patient with clinical history and cerebrospinal fluid findings compatible with the diagnosis of HaNDL in whom Borrelia lusitaniae was identified in cerebrospinal fluid by polymerase chain reaction.

Targeted next generation sequencing identifies novel pathogenic variants and provides molecular diagnoses in a cohort of pediatric and adult patients with unexplained mitochondrial dysfunction.

Mitochondrial diseases (MD) are a group of rare inherited disorders, characterized by phenotypic heterogeneity, with hitherto no effective therapeutic options. The aim of this study was to develop a next generation sequencing (NGS) strategy, by using a custom gene panel and whole mitochondrial genome, to identify the disease causing pathogenic variants in 146 patients suspicious of MD. The molecular analysis of this cohort revealed six novel and 15 described pathogenic variants, as well as 26 variants of unknown significance. Our findings are expanding the mutational landscape of these disorders and support the use of a NGS strategy for a higher diagnostic yield.

Síndrome de Alice no país das maravilhas e reativação de infeção por Epstein-Barr / Alice in wonderland syndrome and reactivation of infection by Epstein-Barr

INTRODUÇÃO: O síndrome de Alice no País das Maravilhas (SAPM) é uma entidade rara que pode ocorre no contexto de várias condições clínicas, sendo a infeção por vírus Epstein-barr (EBV) a mais comum nas causas infeciosas. Apresenta-se um caso de SAPM associado a infeção a EBV alertando para a necessidade de investigação etiológica destes casos. RELATO DE CASO: Criança de 8 anos, com síndrome de Asperger que, no contexto de amigdalite aguda e febre, surgiu com episódios paroxísticos de alguns minutos de metamorfopsias (macro e micropsia), distorção da perceção das vozes e sensação de medo. A ressonância magnética e o eletroencefalograma foram normais, e o exame citoquímico do líquor foi normal mas a polimerase chain reaction (PCR) foi positiva para vírus EBV. As serologias para EBV, repetidas 3 e 10 semanas após a avaliação inicial, confirmaram uma reativação da infeção por este agente. O doente ficou assintomático após 2 semanas e não houve recidivas. CONCLUSÕES: A investigação de metamorfopsias ou síndrome de SAPM é mandatória pois podem indiciar patologia grave, nomeadamente lesão cerebral ou epilepsia focal. Embora a doença seja rara a etiologia infecciosa deve ser excluída mesmo em doentes com perturbação prévia do comportamento.

INTRODUCTION: Alice in wonderland syndrome (AWS) is a rare condition which may occur as a sign of multiple conditions, with the most frequent infectious etiology being Epstein-barr virus (EBV) infection. We present a case of an AWS caused by EBV infection to alert for the need to investigate these cases. CASE REPORT: 8-year-old boy with Asperger syndrome who developed, in the context of tonsillitis and fever, brief paroxystic episodes of metamorfopsias (macro and micropsia), with voice perception distortion and fear. Physical exam was otherwise normal. Brian magnetic resonance and electroencephalogram were normal, liquor cytochemical exam was normal but Epstein-barr virus (EBV) polimerase chain reaction was positive. EBV blood serologies, repeated 3 and 10 weeks after the initial evaluation, confirmed the reactivation of this agent's infection. Symptoms succumbed 2 weeks after its beginning, with no relapses. CONCLUSIONS: Metamorfopsias or AWS impose etiological investigation because they may occur due to severe disease, namely cerebral lesion or focal epilepsy. Although it is a rare disease, infectious causes should be excluded, even in patients with previous disturbed behavior.

Fatal Central Nervous System Lymphocytic Vasculitis after Treatment for Burkitt Lymphoma in a Patient with a SH2D1A Mutation.

Very rarely, patients with X-linked lymphoproliferative syndrome type 1 present central nervous system vasculitis. We report a patient carrying a SH2D1A mutation that, after treatment for lymphoma developed fatal central nervous system vasculitis. He lacked signs of ongoing Epstein-Barr virus infection. We propose that impaired T cell homeostasis caused by SAP deficiency facilitates aberrant CD8 T cell activation against vascular antigens promoting clinical manifestations.

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.

Identification of novel genetic causes of Rett syndrome-like phenotypes.

BACKGROUND: The aim of this work was to identify new genetic causes of Rett-like phenotypes using array comparative genomic hybridisation and a whole exome sequencing approach. METHODS AND RESULTS: We studied a cohort of 19 Portuguese patients (16 girls, 3 boys) with a clinical presentation significantly overlapping Rett syndrome (RTT). Genetic analysis included filtering of the single nucleotide variants and indels with preference for de novo, homozygous/compound heterozygous, or maternally inherited X linked variants. Examination by MRI and muscle biopsies was also performed. Pathogenic genomic imbalances were found in two patients (10.5%): an 18q21.2 deletion encompassing four exons of the TCF4 gene and a mosaic UPD of chromosome 3. Variants in genes previously implicated in neurodevelopmental disorders (NDD) were identified in six patients (32%): de novo variants in EEF1A2, STXBP1 and ZNF238 were found in three patients, maternally inherited X linked variants in SLC35A2, ZFX and SHROOM4 were detected in two male patients and one homozygous variant in EIF2B2 was detected in one patient. Variants were also detected in five novel NDD candidate genes (26%): we identified de novo variants in the RHOBTB2, SMARCA1 and GABBR2 genes; a homozygous variant in EIF4G1; compound heterozygous variant in HTT. CONCLUSIONS: Network analysis reveals that these genes interact by means of protein interactions with each other and with the known RTT genes. These findings expand the phenotypical spectrum of previously known NDD genes to encompass RTT-like clinical presentations and identify new candidate genes for RTT-like phenotypes.

Variant Rett syndrome in a girl with a pericentric X-chromosome inversion leading to epigenetic changes and overexpression of the MECP2 gene.

Rett syndrome is a neurodevelopmental disorder caused by mutations in the MECP2 gene. We investigated the genetic basis of disease in a female patient with a Rett-like clinical. Karyotype analysis revealed a pericentric inversion in the X chromosome -46,X,inv(X)(p22.1q28), with breakpoints in the cytobands where the MECP2 and CDKL5 genes are located. FISH analysis revealed that the MECP2 gene is not dislocated by the inversion. However, and in spite of a balanced pattern of X inactivation, this patient displayed hypomethylation and an overexpression of the MECP2 gene at the mRNA level in the lymphocytes (mean fold change: 2.55±0.38) in comparison to a group of control individuals; the expression of the CDKL5 gene was similar to that of controls (mean fold change: 0.98±0.10). No gains or losses were detected in the breakpoint regions encompassing known or suspected transcription regulatory elements. We propose that the de-regulation of MECP2 expression in this patient may be due to alterations in long-range genomic interactions caused by the inversion and hypothesize that this type of epigenetic de-regulation of the MECP2 may be present in other RTT-like patients.

Anti-N-methyl-D-aspartate receptor encephalitis with positive serum antithyroid antibodies, IgM antibodies against mycoplasma pneumoniae and human herpesvirus 7 PCR in the CSF.

We report the case of a boy with an encephalopathy associated with extrapyramidal and psychiatric symptoms and anti-N-methyl-D-aspartate receptor antibodies. He had positive serum antithyroid antibodies, IgM antibodies against Mycoplasma pneumoniae and human herpesvirus 7 polymerase chain reaction in the cerebrospinal fluid. He was successfully treated with rituximab, after steroids, intravenous immunoglobulin and plasma exchange. The pathophysiology of this disorder may be post-infectious and autoimmune.

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.

Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling.

The type I interferon system is integral to human antiviral immunity. However, inappropriate stimulation or defective negative regulation of this system can lead to inflammatory disease. We sought to determine the molecular basis of genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome and of other undefined neurological and immunological phenotypes also demonstrating an upregulated type I interferon response. We found that heterozygous mutations in the cytosolic double-stranded RNA receptor gene IFIH1 (also called MDA5) cause a spectrum of neuroimmunological features consistently associated with an enhanced interferon state. Cellular and biochemical assays indicate that these mutations confer gain of function such that mutant IFIH1 binds RNA more avidly, leading to increased baseline and ligand-induced interferon signaling. Our results demonstrate that aberrant sensing of nucleic acids can cause immune upregulation.

Sulfite oxidase deficiency--an unusual late and mild presentation.

INTRODUCTION: Sulfite oxidase deficiency (SOD) is an autosomal recessive inherited disease usually presenting in the neonatal period with severe neurological symptoms including seizures, often refractory to anticonvulsant therapy, and a rapidly progressive encephalopathy resembling neonatal hypoxic ischemia, with premature death. Most patients develop dislocated ocular lenses. Later or milder presentations of SOD are being reported with increasing frequency. These presentations include neurological regression with loss of previously acquired milestones or movement disorders. CASE REPORT: We report a four years old girl presenting with intermittent ataxia and uncoordinated limb movements. A similar episode of ataxia had occurred previously, one year before, with complete neurologic recovery and normal developmental milestones. Bilateral lens dislocation had been recently diagnosed. Cranial MRI demonstrated bilateral globus pallidus enhancement. Low homocysteine was found in plasma and Sulfitest(R) was positive. Further investigations led to confirmation of isolated sulfite oxidase deficiency with no enzyme activity detected on skin fibroblasts culture. DISCUSSION: This case illustrates the clinical variability of SOD and it is not only atypical but also seems to be the mildest form described so far. The association of ectopia lentis with a movement disorder, even without psychomotor regression, should prompt us to look for this diagnosis.