Annapurna Poduri

De novo mutations in epileptic encephalopathies

Epileptic encephalopathies are a devastating group of severe childhood epilepsy disorders for which the cause is often unknown. Here we report a screen for de novo mutations in patients with two classical epileptic encephalopathies: infantile spasms (n = 149) and Lennox–Gastaut syndrome (n = 115). We sequenced the exomes of 264 probands, and their parents, and confirmed 329 de novo mutations. A likelihood analysis showed a significant excess of de novo mutations in the ∼4,000 genes that are the most intolerant to functional genetic variation in the human population (P = 2.9 × 10−3). Among these are GABRB3, with de novo mutations in four patients, and ALG13, with the same de novo mutation in two patients; both genes show clear statistical evidence of association with epileptic encephalopathy. Given the relevant site-specific mutation rates, the probabilities of these outcomes occurring by chance are P = 4.1 × 10−10 and P = 7.8 × 10−12, respectively. Other genes with de novo mutations in this cohort include CACNA1A, CHD2, FLNA, GABRA1, GRIN1, GRIN2B, HNRNPU, IQSEC2, MTOR and NEDD4L. Finally, we show that the de novo mutations observed are enriched in specific gene sets including genes regulated by the fragile X protein (P < 10−8), as has been reported previously for autism spectrum disorders.

Single-Neuron Sequencing Analysis of L1 Retrotransposition and Somatic Mutation in the Human Brain

A major unanswered question in neuroscience is whether there exists genomic variability between individual neurons of the brain, contributing to functional diversity or to an unexplained burden of neurological disease. To address this question, we developed a method to amplify genomes of single neurons from human brains. Because recent reports suggest frequent LINE-1 (L1) retrotransposition in human brains, we performed genome-wide L1 insertion profiling of 300 single neurons from cerebral cortex and caudate nucleus of three normal individuals, recovering >80% of germline insertions from single neurons. While we find somatic L1 insertions, we estimate <0.6 unique somatic insertions per neuron, and most neurons lack detectable somatic insertions, suggesting that L1 is not a major generator of neuronal diversity in cortex and caudate. We then genotyped single cortical cells to characterize the mosaicism of a somatic AKT3 mutation identified in a child with hemimegalencephaly. Single-neuron sequencing allows systematic assessment of genomic diversity in the human brain.

Microdeletion/duplication at 15q13.2q13.3 among individuals with features of autism and other neuropsychiatric disorders

Background: Segmental duplications at breakpoints (BP4–BP5) of chromosome 15q13.2q13.3 mediate a recurrent genomic imbalance syndrome associated with mental retardation, epilepsy, and/or electroencephalogram (EEG) abnormalities. Patients: DNA samples from 1445 unrelated patients submitted consecutively for clinical array comparative genomic hybridisation (CGH) testing at Children’s Hospital Boston and DNA samples from 1441 individuals with autism from 751 families in the Autism Genetic Resource Exchange (AGRE) repository. Results: We report the clinical features of five patients with a BP4–BP5 deletion, three with a BP4–BP5 duplication, and two with an overlapping but smaller duplication identified by whole genome high resolution oligonucleotide array CGH. These BP4–BP5 deletion cases exhibit minor dysmorphic features, significant expressive language deficits, and a spectrum of neuropsychiatric impairments that include autism spectrum disorder, attention deficit hyperactivity disorder, anxiety disorder, and mood disorder. Cognitive impairment varied from moderate mental retardation to normal IQ with learning disability. BP4–BP5 covers ∼1.5 Mb (chr15:28.719–30.298 Mb) and includes six reference genes and 1 miRNA gene, while the smaller duplications cover ∼500 kb (chr15:28.902–29.404 Mb) and contain three reference genes and one miRNA gene. The BP4–BP5 deletion and duplication events span CHRNA7, a candidate gene for seizures. However, none of these individuals reported here have epilepsy, although two have an abnormal EEG. Conclusions: The phenotype of chromosome 15q13.2q13.3 BP4–BP5 microdeletion/duplication syndrome may include features of autism spectrum disorder, a variety of neuropsychiatric disorders, and cognitive impairment. Recognition of this broader phenotype has implications for clinical diagnostic testing and efforts to understand the underlying aetiology of this syndrome.

Somatic Mutation, Genomic Variation, and Neurological Disease

Background Genetic mutations that cause human disease are conventionally considered to be inherited from one’s parents and present in all somatic (body) cells. We do know, however, that most mutations that cause cancer arise somatically, and we are becoming increasingly aware of mutations that cause other diseases and that arise de novo, meaning they are undetectable in the parents. Some such de novo mutations arise in the gamete of a parent, but some arise after fertilization during embryonic development, generating somatic mutations. Somatic mutations occur in several neurodevelopmental diseases associated with epilepsy, autism spectrum disorders, and intellectual disability, although their broader relevance for neurological disease is unknown. Cortical development—origins of pyramidal neurons and astrocytes in the cerebral cortex. (A) A neuroepithelial cell (red) at the ventricular zone serves as progenitor for both a pyramidal neuron (green-blue) as well as a radial glial cell (gold). (B) A newly differentiated neuron (blue) migrates along a radial glial process

Using Whole-Exome Sequencing to Identify Inherited Causes of Autism

Despite significant heritability of autism spectrum disorders (ASDs), their extreme genetic heterogeneity has proven challenging for gene discovery. Studies of primarily simplex families have implicated de novo copy number changes and point mutations, but are not optimally designed to identify inherited risk alleles. We apply whole-exome sequencing (WES) to ASD families enriched for inherited causes due to consanguinity and find familial ASD associated with biallelic mutations in disease genes (AMT, PEX7, SYNE1, VPS13B, PAH, and POMGNT1). At least some of these genes show biallelic mutations in nonconsanguineous families as well. These mutations are often only partially disabling or present atypically, with patients lacking diagnostic features of the Mendelian disorders with which these genes are classically associated. Our study shows the utility of WES for identifying specific genetic conditions not clinically suspected and the importance of partial loss of gene function in ASDs.

Somatic Activation of AKT3 Causes Hemispheric Developmental Brain Malformations

Hemimegalencephaly (HMG) is a developmental brain disorder characterized by an enlarged, malformed cerebral hemisphere, typically causing epilepsy that requires surgical resection. We studied resected HMG tissue to test whether the condition might reflect somatic mutations affecting genes critical to brain development. We found that two out of eight HMG samples showed trisomy of chromosome 1q, which encompasses many genes, including AKT3, a gene known to regulate brain size. A third case showed a known activating mutation in AKT3 (c.49G→A, creating p.E17K) that was not present in the patients blood cells. Remarkably, the E17K mutation in AKT3 is exactly paralogous to E17K mutations in AKT1 and AKT2 recently discovered in somatic overgrowth syndromes. We show that AKT3 is the most abundant AKT paralog in the brain during neurogenesis and that phosphorylated AKT is abundant in cortical progenitor cells. Our data suggest that somatic mutations limited to the brain could represent an important cause of complex neurogenetic disease.

Somatic mutations in cerebral cortical malformations.

BACKGROUND Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated. METHODS Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing. RESULTS Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria. CONCLUSIONS Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.).

Single-Cell, Genome-wide Sequencing Identifies Clonal Somatic Copy-Number Variation in the Human Brain

SUMMARY De novo copy-number variants (CNVs) can cause neuropsychiatric disease, but the degree to which they occur somatically, and during development, is unknown. Single-cell whole-genome sequencing (WGS) in >200 single cells, including >160 neurons from three normal and two pathological human brains, sensitively identified germline trisomy of chromosome 18 but found most (≥95%) neurons in normal brain tissue to be euploid. Analysis of a patient with hemimegalencephaly (HMG) due to a somatic CNV of chromosome 1q found unexpected tetrasomy 1q in ~20% of neurons, suggesting that CNVs in a minority of cells can cause widespread brain dysfunction. Single-cell analysis identified large (>1 Mb) clonal CNVs in lymphoblasts and in single neurons from normal human brain tissue, suggesting that some CNVs occur during neurogenesis. Many neurons contained one or more large candidate private CNVs, including one at chromosome 15q13.2-13.3, a site of duplication in neuropsychiatric conditions. Large private and clonal somatic CNVs occur in normal and diseased human brains.

Epilepsy Genetics—Past, Present, and Future

Human epilepsy is a common and heterogeneous condition in which genetics play an important etiological role. We begin by reviewing the past history of epilepsy genetics, a field that has traditionally included studies of pedigrees with epilepsy caused by defects in ion channels and neurotransmitters. We highlight important recent discoveries that have expanded the field beyond the realm of channels and neurotransmitters and that have challenged the notion that single genes produce single disorders. Finally, we project toward an exciting future for epilepsy genetics as large-scale collaborative phenotyping studies come face to face with new technologies in genomic medicine.

Mammalian target of rapamycin pathway mutations cause hemimegalencephaly and focal cortical dysplasia

Focal malformations of cortical development, including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are important causes of intractable childhood epilepsy. Using targeted and exome sequencing on DNA from resected brain samples and nonbrain samples from 53 patients with FCD or HME, we identified pathogenic germline and mosaic mutations in multiple PI3K/AKT pathway genes in 9 patients, and a likely pathogenic variant in 1 additional patient. Our data confirm the association of DEPDC5 with sporadic FCD but also implicate this gene for the first time in HME. Our findings suggest that modulation of the mammalian target of rapamycin pathway may hold promise for malformation‐associated epilepsy. Ann Neurol 2015;77:720–725