Whole exome sequencing in the diagnosis of cerebellar developmental and brain neuronal migration defects

As malformações do sistema nervoso central são a segunda causa mais comum de anomalias congênitas, após as cardiopatias, sendo responsáveis por significativa morbi-mortalidade infantil. No Brasil, cerca de um terço dos óbitos infantis por essa causa correspondem a malformações cerebrais, muitas de etiologia ainda mal definida. O desenvolvimento do cérebro e do cerebelo ocorre em etapas como diferenciação, proliferação, migração e desenvolvimento pós-migracional, orientadas pelas mesmas proteínas do citoesqueleto, razão pela qual é comum a associação, por exemplo, de malformações do desenvolvimento cerebelar com distúrbios da migração neuronal cerebral. O sequenciamento do genoma, ou sua porção codificante, o exoma, tem permitido elucidar a etiologia genética de muitas malformações encefálicas, bem como sugerido novos fenótipos associados a mutações gênicas já descritas. O principal objetivo do presente estudo foi investigar a importância do sequenciamento completo do exoma no esclarecimento etiológico de malformações cerebrais e cerebelares, especificamente dos defeitos do desenvolvimento cerebelar e da migração neuronal cerebral. Trata-se de um estudo observacional de série de casos, descritivo e retrospectivo. Foi analisada uma amostra de 20 pacientes, com idades entre 1 e 12 anos, com defeitos do desenvolvimento cerebelar e da migração neuronal cerebral, submetidos a investigação genética no período de 19/02/2013 a 16/06/2020. Foram avaliados exames de neuroimagem já realizados, e revisados por radiologistas, com a finalidade de padronizar a análise das imagens. A proporção de prevalência entre os sexos foi de 1:1. Todos os pacientes avaliados apresentavam atraso do desenvolvimento neuropsicomotor, sendo epilepsia de difícil controle a segunda manifestação clínica mais comum (75%). Ao exame clínico, hipotonia muscular global foi o achado mais frequente. À análise de neuroimagem, as alterações mais comuns foram hipoplasia pontocerebelar e cerebelar, coexistindo com distúrbios da migração neuronal cortical em apenas um paciente. Dos 16 genes encontrados neste estudo, 5 (31.2%) codificam proteínas relacionadas ao citoesqueleto, sendo que 3 (18.7% do total) codificam subunidades das tubulinas, proteínas dos microtúbulos. Do total de variantes encontradas, 33.3% jamais haviam sido descritas na literatura ou em bancos de dados de variantes. Em um caso, foi encontrada uma variante descrita em gene de lissencefalia (PAFAH1B1), sendo o fenótipo de hipoplasia pontocerebelar sem distúrbio de migração neuronal. Em outro, com fenótipo de polimicrogiria, foi encontrada variante (em ATP1A3) relatada como patogênica na literatura, sem descrição de fenótipo; no entanto, havia relato de malformações corticais associadas ao ATP1A2, que codifica outra subunidade da mesma proteína, sugerindo uma possível expansão do fenótipo associado ao ATP1A3, o que foi confirmado posteriormente com relato na literatura médica. Desta forma, conclui-se que o sequenciamento completo do exoma tem papel fundamental no diagnóstico dos defeitos no desenvolvimento cerebelar e da migração neuronal cerebral, distúrbios por vezes coexistentes, tendo em comum a presença de mutações em genes relacionados ao citoesqueleto, entre outros. A descoberta e o aprimoramento desta técnica tem possibilitado confirmações diagnósticas a partir de correlações genótipo-fenótipo conhecidas, bem como sugerido novas correlações, com expansões de fenótipos associadas a determinados genes e de genótipos possíveis em determinadas doençasMalformations of the central nervous system are the second most common cause of congenital anomalies, after heart disease, and they are responsible for significant infant morbidity and mortality. In Brazil, about a third of infant deaths from this cause corresponds to brain malformations, many of which are still poorly defined. The development of the brain and cerebellum occurs divided into stages such as differentiation, proliferation, migration and postmigrational development, guided by the same cytoskeletal proteins. For this reason, the association, for example, of cerebellar development malformations with disorders of brain neuronal migration is common. The sequencing of the genome, or its coding portion, the exome, has allowed elucidating the genetic etiology of many encephalic malformations, as well as suggesting new phenotypes associated with known gene mutations. The main objective of the present study was to investigate the role of whole exome sequencing in the diagnosis of brain and cerebellar malformations, specifically cerebellar developmental and brain neuronal migration defects. This is an observational case series, descriptive, retrospective study. A sample of 20 patients, aged 1 to 12 years, with cerebellar developmental and cerebral neuronal migration defects who underwent genetic investigation from Feb. 19, 2013 to June 16, 2020 was analyzed. Neuroimaging exams already performed were evaluated, and reviewed by radiologists, in order to standardize the analysis of the images. The prevalence ratio between genders was 1:1. All patients evaluated presented delayed neuropsychomotor development, and difficult-to-control epilepsy was the second most common clinical manifestation (75%).On clinical examination, global muscular hypotonia was the most frequent finding. On neuroimaging analysis, the most common changes were pontocerebellar and cerebellar hypoplasia, coexisting with cortical neuronal migration disorders in only one patient. Of the 16 genes found in this study, 5 (31.2%) encode proteins related to the cytoskeleton, and 3 (18.7% of the total) encode subunits of tubulins, proteins of microtubules. Of the total number of variants found, 33.3% had never been described in the literature or in variant databases. In one case, a variant described in a lissencephaly gene (PAFAH1B1) was found, with a phenotype of pontocerebellar hypoplasia without neuronal migration disorder. In another one, with a polymicrogyria phenotype, a variant (in ATP1A3) reported as pathogenic in the literature was found, with no phenotype description; however, there had been reports of cortical malformations associated with the ATP1A2 gene, which encodes another subunit of the same protein, suggesting a possible expansion of the phenotype associated with the ATP1A3 gene, which could be confirmed later with a report in the medical literature. Thus, we conclude that whole exome sequencing plays a key role in the diagnosis of defects in cerebellar development and brain neuronal migration, disorders that sometimes coexist, having in common the presence of mutations in genes related to the cytoskeleton, among others. The discovery and improvement of this technique has enabled diagnostic confirmation from known genotype-phenotype correlations, as well as suggesting new correlations, with expansions of phenotypes associated with certain genes and of possible genotypes in certain disease

A novel complex neurological phenotype due to a homozygous mutation in FDX2

Defects in iron–sulphur [Fe-S] cluster biogenesis are increasingly recognized as causing neurological disease. Mutations in a number of genes that encode proteins involved in mitochondrial [Fe-S] protein assembly lead to complex neurological phenotypes. One class of proteins essential in the early cluster assembly are ferredoxins. FDX2 is ubiquitously expressed and is essential in the de novo formation of [2Fe-2S] clusters in humans. We describe and genetically define a novel complex neurological syndrome identified in two Brazilian families, with a novel homozygous mutation in FDX2. Patients were clinically evaluated, underwent MRI, nerve conduction studies, EMG and muscle biopsy. To define the genetic aetiology, a combination of homozygosity mapping and whole exome sequencing was performed. We identified six patients from two apparently unrelated families with autosomal recessive inheritance of a complex neurological phenotype involving optic atrophy and nystagmus developing by age 3, followed by myopathy and recurrent episodes of cramps, myalgia and muscle weakness in the first or second decade of life. Sensory-motor axonal neuropathy led to progressive distal weakness. MRI disclosed a reversible or partially reversible leukoencephalopathy. Muscle biopsy demonstrated an unusual pattern of regional succinate dehydrogenase and cytochrome c oxidase deficiency with iron accumulation. The phenotype was mapped in both families to the same homozygous missense mutation in FDX2 (c.431C 4 T, p.P144L). The deleterious effect of the mutation was validated by real-time reverse transcription polymerase chain reaction and western blot analysis, which demonstrated normal expression of FDX2 mRNA but severely reduced expression of FDX2 protein in muscle tissue. This study describes a novel complex neurological phenotype with unusual MRI and muscle biopsy features, conclusively mapped to a mutation in FDX2, which encodes a ubiquitously expressed mitochondrial ferredoxin essential for early [Fe-S] cluster biogenesis