Jacob F. Baranoski

Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration

Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1GLU7ALA), shared by all affected subjects. As demonstrated by isothermal titration calorimetry, purified UCHL1GLU7ALA, compared with WT, exhibited at least sevenfold reduced affinity for ubiquitin. In vitro, the mutation led to a near complete loss of UCHL1 hydrolase activity. The GLU7ALA variant is predicted to interfere with the substrate binding by restricting the proper positioning of the substrate for tunneling underneath the cross-over loop spanning the catalytic cleft of UCHL1. This interference with substrate binding, combined with near complete loss of hydrolase activity, resulted in a >100-fold reduction in the efficiency of UCHL1GLU7ALA relative to WT. These findings demonstrate a broad requirement of UCHL1 in the maintenance of the nervous system.

Seizure control for intracranial arteriovenous malformations is directly related to treatment modality: a meta-analysis

Object Seizures are a common presenting sign of intracranial arteriovenous malformations (AVMs). The object of this meta-analysis was to determine if the modality selected to treat AVMs affects the rate of seizure outcomes. Methods All published data describing seizure status as an outcome goal over the past 20 years were included in this study. Seizure outcomes following microsurgery (MS), endovascular embolization for cure (EVE), or stereotactic radiosurgery (SRS) were compared using a validated random effect logistic regression approach. Results 24 studies, with a total of 1157 patients, were analyzed. Overall, the microsurgical group had the best seizure control (p<0.01), with the relative predicted rates of seizure outcome as follows: MS 78.3% (95% CI 70.1% to 85.8%); SRS 62.8% (95% CI 55.0% to 70.0%); and EVE 49.3% (95% CI 32.1% to 66.6%). Patients in the SRS group who had complete obliteration of their AVMs achieved the highest rate of seizure control (85.2% (95% CI 79.1% to 91.2%); p<0.01). The development of new onset seizures occurred more frequently in patients undergoing EVE (39.4% (95% CI 8.1% to 67.8%)) compared with MS (9.1% (95% CI 5.0% to 13.1%)) and SRS (5.4% (95% CI 3.0% to 7.8%)) (p<0.3 and p<0.01, respectively). Conclusions This is the first meta-analysis designed to study relative rates of seizure outcomes following the currently utilized AVM treatment modalities. In general, MS results in the highest proportion of seizure control. However, if SRS results in successful obliteration of the AVM, then this modality is the most effective in achieving seizure control.

Mutations in KATNB1 Cause Complex Cerebral Malformations by Disrupting Asymmetrically Dividing Neural Progenitors

Exome sequencing analysis of over 2,000 children with complex malformations of cortical development identified five independent (four homozygous and one compound heterozygous) deleterious mutations in KATNB1, encoding the regulatory subunit of the microtubule-severing enzyme Katanin. Mitotic spindle formation is defective in patient-derived fibroblasts, a consequence of disrupted interactions of mutant KATNB1 with KATNA1, the catalytic subunit of Katanin, and other microtubule-associated proteins. Loss of KATNB1 orthologs in zebrafish (katnb1) and flies (kat80) results in microcephaly, recapitulating the human phenotype. In the developing Drosophila optic lobe, kat80 loss specifically affects the asymmetrically dividing neuroblasts, which display supernumerary centrosomes and spindle abnormalities during mitosis, leading to cell cycle progression delays and reduced cell numbers. Furthermore, kat80 depletion results in dendritic arborization defects in sensory and motor neurons, affecting neural architecture. Taken together, we provide insight into the mechanisms by which KATNB1 mutations cause human cerebral cortical malformations, demonstrating its fundamental role during brain development.

Whole-exome sequencing defines the mutational landscape of pheochromocytoma and identifies KMT2D as a recurrently mutated gene.

As subsets of pheochromocytomas (PCCs) lack a defined molecular etiology, we sought to characterize the mutational landscape of PCCs to identify novel gene candidates involved in disease development. A discovery cohort of 15 PCCs wild type for mutations in PCC susceptibility genes underwent whole‐exome sequencing, and an additional 83 PCCs served as a verification cohort for targeted sequencing of candidate mutations. A low rate of nonsilent single nucleotide variants (SNVs) was detected (6.1/sample). Somatic HRAS and EPAS1 mutations were observed in one case each, whereas the remaining 13 cases did not exhibit variants in established PCC genes. SNVs aggregated in apoptosis‐related pathways, and mutations in COSMIC genes not previously reported in PCCs included ZAN, MITF, WDTC1, and CAMTA1. Two somatic mutations and one constitutional variant in the well‐established cancer gene lysine (K)‐specific methyltransferase 2D (KMT2D, MLL2) were discovered in one sample each, prompting KMT2D screening using focused exome‐sequencing in the verification cohort. An additional 11 PCCs displayed KMT2D variants, of which two were recurrent. In total, missense KMT2D variants were found in 14 (11 somatic, two constitutional, one undetermined) of 99 PCCs (14%). Five cases displayed somatic mutations in the functional FYR/SET domains of KMT2D, constituting 36% of all KMT2D‐mutated PCCs. KMT2D expression was upregulated in PCCs compared to normal adrenals, and KMT2D overexpression positively affected cell migration in a PCC cell line. We conclude that KMT2D represents a recurrently mutated gene with potential implication for PCC development.

NGLY1 mutation causes neuromotor impairment, intellectual disability, and neuropathy.

N-glycanase 1 (NGLY1) is a conserved enzyme that is responsible for the deglycosylation of misfolded N-glycosylated proteins in the cytoplasm prior to their proteasome-mediated degradation. Disruption of this degradation process has been associated with various neurologic diseases including amyotrophic lateral sclerosis and Parkinsons disease. Here, we describe two siblings with neuromotor impairment, apparent intellectual disability, corneal opacities, and neuropathy who were found to possess a novel homozygous frame-shift mutation due to a 4 base pair deletion in NGLY1 (c.1533_1536delTCAA, p.Asn511LysfsX51). We hypothesize that this mutation likely limits the capability of neuronal cells to respond to stress due to accumulation of misfolded proteins, thereby impairing their survival and resulting in progressive loss of neurological function.

Paediatric hepatocellular carcinoma due to somatic CTNNB1 and NFE2L2 mutations in the setting of inherited bi-allelic ABCB11 mutations

Hepatocellular carcinoma (HCC) rarely occurs in childhood. We describe a patient with new onset of pruritus at 8 months of age who at 17 months of age was found to have a 2.5 cm HCC. To delineate the possible genetic basis of this tumour, we performed whole exome sequencing (WES) of the germline DNA and identified two novel predictably deleterious missense mutations in ABCB11, encoding bile salt export pump (BSEP), confirmed in the parental DNA as bi-allelic and inherited. Although inherited ABCB11 mutations have previously been linked to HCC in a small number of cases, the molecular mechanisms of hepatocellular carcinogenesis in ABCB11 disease are unknown. WES of the HCC tissue uncovered somatic driver mutations in the beta-catenin (CTNNB1) and nuclear-factor-erythroid-2-related-factor-2 (NFE2L2) genes. Moreover, clonality analysis predicted that the CTNNB1 mutation was clonal and occurred earlier during carcinogenesis, whereas the NFE2L2 mutation was acquired later. Interestingly, background liver parenchyma showed no inflammation or fibrosis and BSEP expression was preserved. This is the first study to identify somatic CTNNB1 and NFE2L2 mutations in early childhood arisen in the setting of inherited bi-allelic ABCB11 mutations. Rapid WES analysis expedited this childs diagnosis and treatment, and likely improved her prognosis.

Construct and face validity of a virtual reality–based camera navigation curriculum

INTRODUCTION Camera handling and navigation are essential skills in laparoscopic surgery. Surgeons rely on camera operators, usually the least experienced members of the team, for visualization of the operative field. Essential skills for camera operators include maintaining orientation, an effective horizon, appropriate zoom control, and a clean lens. Virtual reality (VR) simulation may be a useful adjunct to developing camera skills in a novice population. No standardized VR-based camera navigation curriculum is currently available. We developed and implemented a novel curriculum on the LapSim VR simulator platform for our residents and students. We hypothesize that our curriculum will demonstrate construct and face validity in our trainee population, distinguishing levels of laparoscopic experience as part of a realistic training curriculum. METHODS Overall, 41 participants with various levels of laparoscopic training completed the curriculum. Participants included medical students, surgical residents (Postgraduate Years 1-5), fellows, and attendings. We stratified subjects into three groups (novice, intermediate, and advanced) based on previous laparoscopic experience. We assessed face validity with a questionnaire. The proficiency-based curriculum consists of three modules: camera navigation, coordination, and target visualization using 0° and 30° laparoscopes. Metrics include time, target misses, drift, path length, and tissue contact. We analyzed data using analysis of variance and Students t-test. RESULTS We noted significant differences in repetitions required to complete the curriculum: 41.8 for novices, 21.2 for intermediates, and 11.7 for the advanced group (P < 0.05). In the individual modules, coordination required 13.3 attempts for novices, 4.2 for intermediates, and 1.7 for the advanced group (P < 0.05). Target visualization required 19.3 attempts for novices, 13.2 for intermediates, and 8.2 for the advanced group (P < 0.05). Participants believe that training improves camera handling skills (95%), is relevant to surgery (95%), and is a valid training tool (93%). Graphics (98%) and realism (93%) were highly regarded. CONCLUSIONS The VR-based camera navigation curriculum demonstrates construct and face validity for our training population. Camera navigation simulation may be a valuable tool that can be integrated into training protocols for residents and medical students during their surgery rotations.

Integrated genomic analyses of de novo pathways underlying atypical meningiomas

Meningiomas are mostly benign brain tumours, with a potential for becoming atypical or malignant. On the basis of comprehensive genomic, transcriptomic and epigenomic analyses, we compared benign meningiomas to atypical ones. Here, we show that the majority of primary (de novo) atypical meningiomas display loss of NF2, which co-occurs either with genomic instability or recurrent SMARCB1 mutations. These tumours harbour increased H3K27me3 signal and a hypermethylated phenotype, mainly occupying the polycomb repressive complex 2 (PRC2) binding sites in human embryonic stem cells, thereby phenocopying a more primitive cellular state. Consistent with this observation, atypical meningiomas exhibit upregulation of EZH2, the catalytic subunit of the PRC2 complex, as well as the E2F2 and FOXM1 transcriptional networks. Importantly, these primary atypical meningiomas do not harbour TERT promoter mutations, which have been reported in atypical tumours that progressed from benign ones. Our results establish the genomic landscape of primary atypical meningiomas and potential therapeutic targets.

Brain Malformations Associated With Knobloch Syndrome—Review of Literature, Expanding Clinical Spectrum, and Identification of Novel Mutations

BACKGROUND Knobloch syndrome is a rare, autosomal recessive, developmental disorder characterized by stereotyped ocular abnormalities with or without occipital skull deformities (encephalocele, bone defects, and cutis aplasia). Although there is clear heterogeneity in clinical presentation, central nervous system malformations, aside from the characteristic encephalocele, have not typically been considered a component of the disease phenotype. METHODS Four patients originally presented for genetic evaluation of symptomatic structural brain malformations. Whole-genome genotyping, whole-exome sequencing, and confirmatory Sanger sequencing were performed. Using immunohistochemical analysis, we investigated the protein expression pattern of COL18A1 in the mid-fetal and adult human cerebral cortex and then analyzed the spatial and temporal changes in the expression pattern of COL18A1 during human cortical development using the Human Brain Transcriptome database. RESULTS We identified two novel homozygous deleterious frame-shift mutations in the COL18A1 gene. On further investigation of these patients and their families, we found that many exhibited certain characteristics of Knobloch syndrome, including pronounced ocular defects. Our data strongly support an important role for COL18A1 in brain development, and this report contributes to an enhanced characterization of the brain malformations that can result from deficiencies of collagen XVIII. CONCLUSIONS This case series highlights the diagnostic power and clinical utility of whole-exome sequencing technology-allowing clinicians and physician scientists to better understand the pathophysiology and presentations of rare diseases. We suggest that patients who are clinically diagnosed with Knobloch syndrome and/or found to have COL18A1 mutations via genetic screening should be investigated for potential structural brain abnormalities even in the absence of an encephalocele.

Whole-exome sequencing identified a patient with TMCO1 defect syndrome and expands the phenotic spectrum

To the Editor : In 2010, Xin et al. described a new autosomal-recessive syndrome which includes mental retardation, dysmorphism, skeletal and neurological findings in an Amish family secondary to mutations in the Transmembrane and coiled-coil domain-containing protein 1 (TMCO1 ) gene (1). Here, we report the first non-Amish case with a TMCO1 mutation. Our index case is a 7-year-old boy, SA (Fig. 1a, IV-3), who presented to paediatric neurology clinic due to delayed development (Fig. S1, Supporting information). His parents were first-cousins (Fig. 1a) and he was born at 37 weeks gestation with a head circumference of 38 cm (90%). SA was hypotonic at birth with feeding difficulties and was diagnosed with hypothyroidism. He later was found to have significant delays with the neurodevelopmental milestones, being unable to walk and with minimal speech. His family noted him to be anxious and showing selfmutilating behavior like chewing his fingers. When he was examined at the referring paediatric neurology clinic at 7 years of age, his head circumference was 53 cm (75–90%), his weight was 17 kg, (<5%) and height 113 cm (5%). His general physical examination was remarkable for several dysmorphic features including short neck, low hairline, low set ears, synophrys, hypertelorism, antevert nares, high-arched palate, prognatism, hyperextensbile fingers, pectus carinatum, scoliosis and genu varus. On neurological examination, he was awake but unable to speak except a few basic words. He was unable to walk, feed himself or perform activities of daily living. There were no abnormalities in routine laboratory tests, including complete blood count (CBC), blood chemistries, urine test, and plasma aminoacid levels. Chest X-ray was remarkable for rib and scapula abnormalities. On brain magnetic resonance imaging, dysgenesis of the corpus callosum and cerebellar herniation were detected (Fig. S2). In order to identify the disease causing variant, we performed homozygosity mapping followed by whole exome sequencing using Nimblegen solid phase arrays (Roche NimbleGen, Inc., Madison, WI) and the Illumina HiSeq 2000 (Illumina, Inc., San Diego, CA). We achieved a mean coverage of 30.5×, and 91.47% of all targeted bases were covered more than four times, sufficient to identify novel homozygous variants with high specificity (2) (Tables S1 and S2). We a)