Evaluation of Presumably Disease Causing SCN1A Variants in a Cohort of Common Epilepsy Syndromes

in PLoS ONE (2016)

Objective The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing ... [more ▼]

Objective The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing, clinical laboratories are generating an ever-increasing catalogue of SCN1A variants. Variants are more likely to be classified as pathogenic if they have already been identified previously in a patient with epilepsy. Here, we critically re-evaluate the pathogenicity of this class of variants in a cohort of patients with common epilepsy syndromes and subsequently ask whether a significant fraction of benign variants have been misclassified as pathogenic. Methods We screened a discovery cohort of 448 patients with a broad range of common genetic epilepsies and 734 controls for previously reported SCN1A mutations that were assumed to be disease causing. We re-evaluated the evidence for pathogenicity of the identified variants using in silico predictions, segregation, original reports, available functional data and assessment of allele frequencies in healthy individuals as well as in a follow up cohort of 777 patients. Results and Interpretation We identified 8 known missense mutations, previously reported as pathogenic, in a total of 17 unrelated epilepsy patients (17/448; 3.80%). Our re-evaluation indicates that 7 out of these 8 variants (p.R27T; p.R28C; p.R542Q; p.R604H; p.T1250M; p.E1308D; p.R1928G; NP_001159435.1) are not pathogenic. Only the p.T1174S mutation may be considered as a genetic risk factor for epilepsy of small effect size based on the enrichment in patients (P = 6.60 x 10−4; OR = 0.32, fishers exact test), previous functional studies but incomplete penetrance. Thus, incorporation of previous studies in genetic counseling of SCN1A sequencing results is challenging and may produce incorrect conclusions. [less ▲]

Identification, recovery, and refinement of hitherto undescribed population-level genomes from the human gastrointestinal tract

in Frontiers in Microbiology (2016), 7(884),

Linking taxonomic identity and functional potential at the population-level is important for the study of mixed microbial communities and is greatly facilitated by the availability of microbial reference ... [more ▼]

Linking taxonomic identity and functional potential at the population-level is important for the study of mixed microbial communities and is greatly facilitated by the availability of microbial reference genomes. While the culture-independent recovery of population-level genomes from environmental samples using the binning of metagenomic data has expanded available reference genome catalogs, several microbial lineages remain underrepresented. Here, we present two reference-independent approaches for the identification, recovery, and refinement of hitherto undescribed population-level genomes. The first approach is aimed at genome recovery of varied taxa and involves multi-sample automated binning using CANOPY CLUSTERING complemented by visualization and human-augmented binning using VIZBINpost hoc. The second approach is particularly well-suited for the study of specific taxa and employs VIZBINde novo. Using these approaches, we reconstructed a total of six population-level genomes of distinct and divergent representatives of the Alphaproteobacteria class, the Mollicutes class, the Clostridiales order, and the Melainabacteria class from human gastrointestinal tract-derived metagenomic data. Our results demonstrate that, while automated binning approaches provide great potential for large-scale studies of mixed microbial communities, these approaches should be complemented with informative visualizations because expert-driven inspection and refinements are critical for the recovery of high-quality population-level genomes. [less ▲]

Integrated multi-omics of the human gut microbiome in a case study of familial type 1 diabetes.

in Nature Microbiology (2016), 2

The gastrointestinal microbiome is a complex ecosystem with functions that shape human health. Studying the relationship between taxonomic alterations and functional repercussions linked to disease ... [more ▼]

The gastrointestinal microbiome is a complex ecosystem with functions that shape human health. Studying the relationship between taxonomic alterations and functional repercussions linked to disease remains challenging. Here, we present an integrative approach to resolve the taxonomic and functional attributes of gastrointestinal microbiota at the metagenomic, metatranscriptomic and metaproteomic levels. We apply our methods to samples from four families with multiple cases of type 1 diabetes mellitus (T1DM). Analysis of intra- and inter-individual variation demonstrates that family membership has a pronounced effect on the structural and functional composition of the gastrointestinal microbiome. In the context of T1DM, consistent taxonomic differences were absent across families, but certain human exocrine pancreatic proteins were found at lower levels. The associated microbial functional signatures were linked to metabolic traits in distinct taxa. The methodologies and results provide a foundation for future large-scale integrated multi-omic analyses of the gastrointestinal microbiome in the context of host-microbe interactions in human health and disease. [less ▲]

A novel Fanconi anemia subtype associated with a dominant-negative mutation in RAD51

in Nature Communications (2015), 6(8829),

Fanconi anemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong ... [more ▼]

Fanconi anemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, “FA-R”, which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and pediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility. [less ▲]

Recessive mutations in SLC13A5 result in a loss of citrate transport and cause neonatal epilepsy, developmental delay and teeth hypoplasia

in Brain : A Journal of Neurology (2015)

The epileptic encephalopathies are a clinically and aetiologically heterogeneous subgroup of epilepsy syndromes. Most epileptic encephalopathies have a genetic cause and patients are often found to carry ... [more ▼]

The epileptic encephalopathies are a clinically and aetiologically heterogeneous subgroup of epilepsy syndromes. Most epileptic encephalopathies have a genetic cause and patients are often found to carry a heterozygous de novo mutation in one of the genes associated with the disease entity. Occasionally recessive mutations are identified: a recent publication described a distinct neonatal epileptic encephalopathy (MIM 615905) caused by autosomal recessive mutations in the SLC13A5 gene. Here, we report eight additional patients belonging to four different families with autosomal recessive mutations in SLC13A5. SLC13A5 encodes a high affinity sodium-dependent citrate transporter, which is expressed in the brain. Neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates; therefore they rely on the uptake of intermediates, such as citrate, to maintain their energy status and neurotransmitter production. The effect of all seven identified mutations (two premature stops and five amino acid substitutions) was studied in vitro, using immunocytochemistry, selective western blot and mass spectrometry. We hereby demonstrate that cells expressing mutant sodium-dependent citrate transporter have a complete loss of citrate uptake due to various cellular loss-of-function mechanisms. In addition, we provide independent proof of the involvement of autosomal recessive SLC13A5 mutations in the development of neonatal epileptic encephalopathies, and highlight teeth hypoplasia as a possible indicator for SLC13A5 screening. All three patients who tried the ketogenic diet responded well to this treatment, and future studies will allow us to ascertain whether this is a recurrent feature in this severe disorder. [less ▲]

De novo loss- or gain-of-function mutations in KCNA2 cause epileptic encephalopathy

in Nature Genetics (2015), 47(4), 393-9

Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features1–6. Using next ... [more ▼]

Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features1–6. Using next-generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium channel KV1.2, in six patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild to moderate intellectual disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype showed almost complete loss of function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype. They carried mutations inducing a drastic gain-of-function effect leading to permanently open channels. These results establish KCNA2 as a new gene involved in human neurodevelopmental disorders through two different mechanisms, predicting either hyperexcitability or electrical silencing of KV1.2-expressing neurons. [less ▲]

Integrated omics analyses of the human gut microbiome in a multiplex family study of type 1 diabetes mellitus.

Poster (2015, March)

The phenotypic spectrum of SCN8A encephalopathy

in Neurology (2015), 84(5), 480-489

Objective: SCN8A encodes the sodium channel voltage-gated α8-subunit (Nav1.6). SCN8A mutations have recently been associated with epilepsy and neurodevelopmental disorders. We aimed to delineate the ... [more ▼]

Objective: SCN8A encodes the sodium channel voltage-gated α8-subunit (Nav1.6). SCN8A mutations have recently been associated with epilepsy and neurodevelopmental disorders. We aimed to delineate the phenotype associated with SCN8A mutations. Methods: We used high-throughput sequence analysis of the SCN8A gene in 683 patients with a range of epileptic encephalopathies. In addition, we ascertained cases with SCN8A mutations from other centers. A detailed clinical history was obtained together with a review of EEG and imaging data. Results: Seventeen patients with de novo heterozygous mutations of SCN8A were studied. Seizure onset occurred at a mean age of 5 months (range: 1 day to 18 months); in general, seizures were not triggered by fever. Fifteen of 17 patients had multiple seizure types including focal, tonic, clonic, myoclonic and absence seizures, and epileptic spasms; seizures were refractory to antiepileptic therapy. Development was normal in 12 patients and slowed after seizure onset, often with regression; 5 patients had delayed development from birth. All patients developed intellectual disability, ranging from mild to severe. Motor manifestations were prominent including hypotonia, dystonia, hyperreflexia, and ataxia. EEG findings comprised moderate to severe background slowing with focal or multifocal epileptiform discharges. Conclusion: SCN8A encephalopathy presents in infancy with multiple seizure types including focal seizures and spasms in some cases. Outcome is often poor and includes hypotonia and movement disorders. The majority of mutations arise de novo, although we observed a single case of somatic mosaicism in an unaffected parent. [less ▲]

Recessive loss-of-function mutations in AP4S1 cause mild fever-sensitive seizures, developmental delay and spastic paraplegia through loss of AP-4 complex assembly

in Human Molecular Genetics (2015), 24(8), 2218-2227

We report two siblings with infantile onset seizures, severe developmental delay and spastic paraplegia, in whom whole genome sequencing revealed compound heterozygous mutations in the AP4S1 gene ... [more ▼]

We report two siblings with infantile onset seizures, severe developmental delay and spastic paraplegia, in whom whole genome sequencing revealed compound heterozygous mutations in the AP4S1 gene, encoding the sigma subunit of the adaptor protein complex 4 (AP-4). The effect of the predicted loss-of-function variants (p.Gln46Profs*9 and p.Arg97*) was further investigated in a patient's fibroblast cell line. We show that the premature stop mutations in AP4S1 result in a reduction of all AP-4 subunits and loss of AP-4 complex assembly. Recruitment of the AP-4 accessory protein, tepsin, to the membrane was also abolished. In retrospect, the clinical phenotype in the family is consistent with previous reports of the AP-4 deficiency syndrome. Our study reports the second family with mutations in AP4S1 and describes the first two patients with loss of AP4S1 and seizures. We further discuss seizure phenotypes in reported patients, highlighting that seizures are part of the clinical manifestation of the AP4-deficiency syndrome. We also hypothesize that endosomal trafficking is a common theme between heritable spastic paraplegia and some inherited epilepsies. [less ▲]

Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes

in Nature Genetics (2014), 46(12), 1327-32

Febrile seizures affect 2–4% of all children1 and have a strong genetic component2. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2)3, 4, 5 have been identified that cause febrile ... [more ▼]

Febrile seizures affect 2–4% of all children1 and have a strong genetic component2. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2)3, 4, 5 have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B6, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees7, 8 identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes. [less ▲]