Greta Gillies

Familial cortical dysplasia caused by mutation in the mammalian target of rapamycin regulator NPRL3

We describe first cousin sibling pairs with focal epilepsy, one of each pair having focal cortical dysplasia (FCD) IIa. Linkage analysis and whole‐exome sequencing identified a heterozygous germline frameshift mutation in the gene encoding nitrogen permease regulator‐like 3 (NPRL3). NPRL3 is a component of GAP Activity Towards Rags 1, a negative regulator of the mammalian target of rapamycin complex 1 signaling pathway. Immunostaining of resected brain tissue demonstrated mammalian target of rapamycin activation. Screening of 52 unrelated individuals with FCD identified 2 additional patients with FCDIIa and germline NPRL3 mutations. Similar to DEPDC5, NPRL3 mutations may be considered as causal variants in patients with FCD or magnetic resonance imaging–negative focal epilepsy. ANN NEUROL 2016;79:132–137

Familial cortical dysplasia type IIA caused by a germline mutation in DEPDC5

Whole‐exome sequencing of two brothers with drug‐resistant, early‐onset, focal epilepsy secondary to extensive type IIA focal cortical dysplasia identified a paternally inherited, nonsense variant of DEPDC5 (c.C1663T, p.Arg555*). This variant has previously been reported to cause familial focal epilepsy with variable foci in patients with normal brain imaging. Immunostaining of resected brain tissue from both brothers demonstrated mammalian target of rapamycin (mTOR) activation. This report shows the histopathological features of cortical dysplasia associated with a DEPDC5 mutation, confirms mTOR dysregulation in the malformed tissue and expands the spectrum of neurological manifestations of DEPDC5 mutations to include severe phenotypes with large areas of cortical malformation.

Familial cortical dysplasia caused by mutation in the mTOR regulator NPRL3

We describe first cousin sibling pairs with focal epilepsy, one of each pair having focal cortical dysplasia (FCD) IIa. Linkage analysis and whole‐exome sequencing identified a heterozygous germline frameshift mutation in the gene encoding nitrogen permease regulator‐like 3 (NPRL3). NPRL3 is a component of GAP Activity Towards Rags 1, a negative regulator of the mammalian target of rapamycin complex 1 signaling pathway. Immunostaining of resected brain tissue demonstrated mammalian target of rapamycin activation. Screening of 52 unrelated individuals with FCD identified 2 additional patients with FCDIIa and germline NPRL3 mutations. Similar to DEPDC5, NPRL3 mutations may be considered as causal variants in patients with FCD or magnetic resonance imaging–negative focal epilepsy. ANN NEUROL 2016;79:132–137

Hemispheric cortical dysplasia secondary to a mosaic somatic mutation in MTOR

Objective: To define causative somatic mutations in resected brain tissue from an infant with intractable epilepsy secondary to hemispheric cortical dysplasia. Methods: Whole-exome sequencing was conducted on genomic DNA derived from both resected brain tissue and peripheral blood leukocytes. Comparison of the brain vs blood sequencing results was performed using bioinformatic methods designed to detect low-frequency genetic variation between tissue pairs. Results: Histopathology of the resected tissue showed dyslamination and dysmorphic neurons, but no balloon cells, consistent with focal cortical dysplasia type IIa. mTOR activation was observed by immunohistochemistry in the dysplasia. A missense mutation (c.4487T>G; p.W1456G) was detected in the FAT domain of MTOR in DNA from the dysplasia but not in lymphocytes. The mutation is predicted damaging (i.e., leading to mTOR activation) and was observed as a low-level mosaic with 8% of cells being heterozygous for the variant. Conclusions: We report the novel finding of an MTOR mutation associated with nonsyndromic cortical dysplasia. Somatic-specific mutations in MTOR and related genes should be considered in a broader spectrum of patients with hemispheric malformations and more restricted forms of cortical dysplasia.

Mutations in DCC cause isolated agenesis of the corpus callosum with incomplete penetrance

Brain malformations involving the corpus callosum are common in children with developmental disabilities. We identified DCC mutations in four families and five sporadic individuals with isolated agenesis of the corpus callosum (ACC) without intellectual disability. DCC mutations result in variable dominant phenotypes with decreased penetrance, including mirror movements and ACC associated with a favorable developmental prognosis. Possible phenotypic modifiers include the type and location of mutation and the sex of the individual.

Expanding the phenotypic spectrum of ARID1B-mediated disorders and identification of altered cell-cycle dynamics due to ARID1B haploinsufficiency.

BackgroundMutations in genes encoding components of the Brahma-associated factor (BAF) chromatin remodeling complex have recently been shown to contribute to multiple syndromes characterised by developmental delay and intellectual disability. ARID1B mutations have been identified as the predominant cause of Coffin-Siris syndrome and have also been shown to be a frequent cause of nonsyndromic intellectual disability. Here, we investigate the molecular basis of a patient with an overlapping but distinctive phenotype of intellectual disability, plantar fat pads and facial dysmorphism.Methods/resultsHigh density microarray analysis of the patient demonstrated a heterozygous deletion at 6q25.3, which resulted in the loss of four genes including AT Rich Interactive Domain 1B (ARID1B). Subsequent quantitative real-time PCR analysis revealed ARID1B haploinsufficiency in the patient. Analysis of both patient-derived and ARID1B knockdown fibroblasts after serum starvation demonstrated delayed cell cycle re-entry associated with reduced cell number in the S1 phase. Based on the patient’s distinctive phenotype, we ascertained four additional patients and identified heterozygous de novo ARID1B frameshift or nonsense mutations in all of them.ConclusionsThis study broadens the spectrum of ARID1B associated phenotypes by describing a distinctive phenotype including plantar fat pads but lacking the hypertrichosis or fifth nail hypoplasia associated with Coffin-Siris syndrome. We present the first direct evidence in patient-derived cells that alterations in cell cycle contribute to the underlying pathogenesis of syndromes associated with ARID1B haploinsufficiency.

Identification of a Novel RNF213 Variant in a Family with Heterogeneous Intracerebral Vasculopathy

Moyamoya disease (MMD) is characterized by progressive occlusion of the internal carotid arteries and their distal branches. Genetic heterogeneity is apparent with multiple loci mapped, and mutations in the ring finger 213 gene (RNF213) were recently identified in typical MMD (1) and non-MMD arterial stenosis/occlusion (2). Understanding of the disease pathogenesis associated with RNF213 mutations remains incomplete, particularly in patients of European ancestry. In our study ‘Identification of a novel RNF213 variant in a family with heterogeneous intracerebral vasculopathy’, we identified a three-generation family of European ancestry with intracerebral vasculopathy displaying variability in age of onset and clinical severity (Fig. 1). None had syndromic features such as neurofibromatosis. The proband (MM1-3) presented at six years with left facial droop and slurred speech. Magnetic resonance imaging (MRI) showed multiple areas of cerebral infarction of different ages, and magnetic resonance angiography (MRA) and conventional angiography (CA) showed a vasculopathy consistent with MMD (Fig. 2). Her brother (MM1-4) presented at five years with two transient ischaemic attacks (TIAs) over 12 h manifest by episodes of right facial droop and right leg weakness. Brain MRI/MRA and CA showed bilateral anterior circulation steno-occlusive vasculopathy without infarction or collaterals. He continued to have TIAs and progressive ischemic changes in the left frontal lobe until two multiple burr hole revascularization procedures were completed. The proband’s 38yo mother (MM1-1) had a long history of ‘hemiplegic migraines’. MRI/ MRA and CA identified bilateral anterior circulation steno-occlusive vasculopathy with basal collaterals, but no ischemic changes. An asymptomatic 40-year-old uncle (MM1-6) showed unilateral cerebral vasculopathy without infarction on MRI/MRA and CA. We performed linkage analysis to prioritize the 20 738 variants identified by exome sequencing of two affected siblings. Only one of 26 linkage regions coincided with a known MMD locus. A heterozygous c.12554 A>C (p.K4185T) transversion in RNF213 (NM_001256071.1) was identified in both siblings and subsequently all affected family members (Fig. 1). K4185T is an excellent candidate as it is predicted damaging by SIFT, is not observed in normative databases, and the highly conserved lysine is located in the C-terminal region where MMD mutations cluster (1). The variant was observed in the clinically and radiologically unaffected maternal grandmother (MM1-15), suggesting incomplete penetrance or somatic mosaicism. The first cousin (MM1-10) carried the variant but displayed no clinical or MRI/MRA abnormalities when screened at age 3.8 years, highlighting the clinical difficulties in interpreting mutations in susceptibility genes. He is considered at risk of developing intracerebral vasculopathy, and follow-up is ongoing. In conclusion, we demonstrate that combining linkage analysis and massively parallel sequencing is an effective strategy to investigate the molecular basis of neurological disorders characterized by genetic variability. Our findings suggest that MMD may lie at the severe end of a spectrum of occlusive intracranial vasculopathies with shared etiologies, and analysis of RNF213 in additional cohorts with cerebral vasculopathies is required.

Heterozygous mutations in HSD17B4 cause juvenile peroxisomal D-bifunctional protein deficiency

Objective: To determine the genetic cause of slowly progressive cerebellar ataxia, sensorineural deafness, and hypergonadotropic hypogonadism in 5 patients from 3 different families. Methods: The patients comprised 2 sib pairs and 1 sporadic patient. Clinical assessment included history, physical examination, and brain MRI. Linkage analysis was performed separately on the 2 sets of sib pairs using single nucleotide polymorphism microarrays, followed by analysis of the intersection of the regions. Exome sequencing was performed on 1 affected patient with variant filtering and prioritization undertaken using these intersected regions. Results: Using a combination of sequencing technologies, we identified compound heterozygous mutations in HSD17B4 in all 5 affected patients. In all 3 families, peroxisomal D-bifunctional protein (DBP) deficiency was caused by compound heterozygosity for 1 nonsense/deletion mutation and 1 missense mutation. Conclusions: We describe 5 patients with juvenile DBP deficiency from 3 different families, bringing the total number of reported patients to 14, from 8 families. This report broadens and consolidates the phenotype associated with juvenile DBP deficiency.

Rasmussen encephalitis tissue transfer program

To the Editors: Rasmussen encephalitis (RE) was first described in 1958 by Theodore Rasmussen and colleagues at the Montreal Neurological Institute. RE is a very rare neuroinflammatory disease characterized by intractable seizures and progressive unilateral neurologic deficits. For most diagnosed cases, resection or disconnection of the affected cerebral hemisphere is the only effective treatment. The extreme rarity of the disease has hampered efforts to understand the cause of RE and to develop alternative nonsurgical treatments. Usually only one or two RE cases may been seen annually at a pediatric epilepsy surgery center, thus it may take many years to accrue enough surgical specimens for research studies, especially those involving modern molecular techniques. In 2011, The RE Children’s Project (www.REChildrens.org), a nonprofit organization founded to increase awareness of the disease and support research focused on finding a cure, brought together investigators from around the world to launch the RE Children’s Research Consortium and discuss ways to accelerate the pace of RE research. With the support of the RE Children’s Project, researchers at Johns Hopkins School of Medicine and David Geffen School of Medicine at UCLA launched an international Tissue Transfer Program and data bank to speed up the pace of RE research. The goal of the Tissue Transfer Program is to collect RE surgical specimens from epilepsy centers around the world and to make biologic samples and clinical data available for RE research. Between 2011 and 2012, the program was initiated with coordination managed at Johns Hopkins and in 2013 the role was transferred to UCLA. A central repository for the collected samples was established within the Rare Epilepsies and Brain Disease Tissue Bank in the Department of Neurosurgery at UCLA (http://neurosurgery.ucla.edu/rareepilepsies-tissue-bank). Excess material from a planned epilepsy surgery that would otherwise be discarded, per institutional guidelines, would be collected for this purpose. All logistics would be handled by the Rare Epilepsies and Brain Disease Tissue Bank Coordinator at UCLA, who would liaise with donor institutions to facilitate the transfer of surgical specimens at no cost to the participating institution. Institutional review board (IRB) approval was obtained to collect surgical specimens from outside institutions, and to distribute material to other centers with institutionally approved research studies. Since the inception of the Tissue Transfer Program, RE specimens have been collected from 33 surgeries at 19 epilepsy centers in six countries (Fig. 1). Stored specimens include both fixed and frozen brain tissue, cerebrospinal fluid, whole blood, plasma, purified peripheral blood mononuclear cells, and brain-infiltrating lymphocytes. With increased awareness of this program, we hope that the pace of RE research will be accelerated. Epilepsy surgery centers around the world are invited to contribute samples and participate in this international effort to cure RE. Applications to access the RE repository should be directed to the Rare Epilepsies and Brain Disease Tissue Bank Coordinator at UCLA (http://neurosurgery.ucla. edu/rare-epilepsies-tissue-bank-contact-us). The Scientific Advisory Board of the RE Children’s Project will review applications for research studies to ensure a fair and rigorous assessment of the proposed work. In addition to RE, the Rare Epilepsies and Brain Disease Tissue bank is actively collecting specimens from other rare or uncommon pediatric epilepsy surgery cases including hemimegalencephaly (HME), focal cortical dysplasia (FCD), and tuberous sclerosis (TSC). For more information visit http://neurosurgery.ucla.edu/rare-epilepsies-tissue-bank.

Somatic GNAQ mutation in the forme fruste of Sturge-Weber syndrome

Objective To determine whether the GNAQ R183Q mutation is present in the forme fruste cases of Sturge-Weber syndrome (SWS) to establish a definitive molecular diagnosis. Methods We used sensitive droplet digital PCR (ddPCR) to detect and quantify the GNAQ mutation in tissues from epilepsy surgery in 4 patients with leptomeningeal angiomatosis; none had ocular or cutaneous manifestations. Results Low levels of the GNAQ mutation were detected in the brain tissue of all 4 cases—ranging from 0.42% to 7.1% frequency—but not in blood-derived DNA. Molecular evaluation confirmed the diagnosis in 1 case in which the radiologic and pathologic data were equivocal. Conclusions We detected the mutation at low levels, consistent with mosaicism in the brain or skin (1.0%–18.1%) of classic cases. Our data confirm that the forme fruste is part of the spectrum of SWS, with the same molecular mechanism as the classic disease and that ddPCR is helpful where conventional diagnosis is uncertain.