Matt De Both

Association of SNPs in EGR3 and ARC with Schizophrenia Supports a Biological Pathway for Schizophrenia Risk

We have previously hypothesized a biological pathway of activity-dependent synaptic plasticity proteins that addresses the dual genetic and environmental contributions to schizophrenia. Accordingly, variations in the immediate early gene EGR3, and its target ARC, should influence schizophrenia susceptibility. We used a pooled Next-Generation Sequencing approach to identify variants across these genes in U.S. populations of European (EU) and African (AA) descent. Three EGR3 and one ARC SNP were selected and genotyped for validation, and three SNPs were tested for association in a replication cohort. In the EU group of 386 schizophrenia cases and 150 controls EGR3 SNP rs1877670 and ARC SNP rs35900184 showed significant associations (p = 0.0078 and p = 0.0275, respectively). In the AA group of 185 cases and 50 controls, only the ARC SNP revealed significant association (p = 0.0448). The ARC SNP did not show association in the Han Chinese (CH) population. However, combining the EU, AA, and CH groups revealed a highly significant association of ARC SNP rs35900184 (p = 2.353 x 10−7; OR [95% CI] = 1.54 [1.310–1.820]). These findings support previously reported associations between EGR3 and schizophrenia. Moreover, this is the first report associating an ARC SNP with schizophrenia and supports recent large-scale GWAS findings implicating the ARC complex in schizophrenia risk. These results support the need for further investigation of the proposed pathway of environmentally responsive, synaptic plasticity-related, schizophrenia genes.

A Frame-Shift Mutation in CAV1 Is Associated with a Severe Neonatal Progeroid and Lipodystrophy Syndrome.

A 3-year-old female patient presenting with an unknown syndrome of a neonatal progeroid appearance, lipodystrophy, pulmonary hypertension, cutis marmorata, feeding disorder and failure to thrive was investigated by whole-genome sequencing. This revealed a de novo, heterozygous, frame-shift mutation in the Caveolin1 gene (CAV1) (p.Phe160X). Mutations in CAV1, encoding the main component of the caveolae in plasma membranes, cause Berardinelli-Seip congenital lipodystrophy type 3 (BSCL). Although BSCL is recessive, heterozygous carriers either show a reduced phenotype of partial lipodystrophy, pulmonary hypertension, or no phenotype. To investigate the pathogenic mechanisms underlying this syndrome in more depth, we performed next generation RNA sequencing of peripheral blood, which showed several dysregulated pathways in the patient that might be related to the phenotypic progeroid features (apoptosis, DNA repair/replication, mitochondrial). Secondly, we found a significant down-regulation of known Cav1 interaction partners, verifying the dysfunction of CAV1. Other known progeroid genes and lipodystrophy genes were also dysregulated. Next, western blotting of lysates of cultured fibroblasts showed that the patient shows a significantly decreased expression of wild-type CAV1 protein, demonstrating a loss-of-function mutation, though her phenotype is more severe that other heterozygotes with similar mutations. This phenotypic variety could be explained by differences in genetic background. Indications for this are supported by additional rare variants we found in AGPAT2 and LPIN1 lipodystrophy genes. CAV1, AGPAT2 and LPIN1 all play an important role in triacylglycerol (TAG) biosynthesis in adipose tissue, and the defective function in different parts of this pathway, though not all to the same extend, could contribute to a more severe lipoatrophic phenotype in this patient. In conclusion, we report, for the first time, an association of CAV1 dysfunction with a syndrome of severe premature aging and lipodystrophy. This may contribute to a better understanding of the aging process and pathogenic mechanisms that contribute to premature aging.

A De Novo Mutation in TEAD1 Causes Non–X-Linked Aicardi Syndrome

PURPOSE Aicardi syndrome (AIC) is a congenital neurodevelopmental disorder characterized by infantile spasms, agenesis of the corpus callosum, and chorioretinal lacunae. Variation in phenotype and disease severity is well documented, but chorioretinal lacunae represent the most constant pathological feature. Aicardi syndrome is believed to be an X-linked-dominant disorder occurring almost exclusively in females, although 46, XY males with AIC have been described. The purpose of this study is to identify genetic factors and pathways involved in AIC. METHODS We performed exome/genome sequencing of 10 children diagnosed with AIC and their parents and performed RNA sequencing on blood samples from nine cases, their parents, and unrelated controls. RESULTS We identified a de novo mutation in autosomal gene TEAD1, expressed in the retina and brain, in a patient with AIC. Mutations in TEAD1 have previously been associated with Sveinssons chorioretinal atrophy, characterized by chorioretinal degeneration. This demonstrates that TEAD1 mutations can lead to different chorioretinal complications. In addition, we found that altered expression of genes associated with synaptic plasticity, neuronal development, retinal development, and cell cycle control/apoptosis is an important underlying potential pathogenic mechanism shared among cases. Last, we found a case with skewed X inactivation, supporting the idea that nonrandom X inactivation might be important in AIC. CONCLUSIONS We expand the phenotype of TEAD1 mutations, demonstrate its importance in chorioretinal complications, and propose the first putative pathogenic mechanisms underlying AIC. Our data suggest that AIC is a genetically heterogeneous disease and is not restricted to the X chromosome, and that TEAD1 mutations may be present in male patients.

Case Report: Novel mutations in TBC1D24 are associated with autosomal dominant tonic-clonic and myoclonic epilepsy and recessive Parkinsonism, psychosis, and intellectual disability

Mutations disrupting presynaptic protein TBC1D24 are associated with a variable neurological phenotype, including DOORS syndrome, myoclonic epilepsy, early-infantile epileptic encephalopathy, and non-syndromic hearing loss. In this report, we describe a family segregating autosomal dominant epilepsy, and a 37-year-old Caucasian female with a severe neurological phenotype including epilepsy, Parkinsonism, psychosis, visual and auditory hallucinations, gait ataxia and intellectual disability. Whole exome sequencing revealed two missense mutations in the TBC1D24 gene segregating within this family (c.1078C>T; p.Arg360Cys and c.404C>T; p.Pro135Leu). The female proband who presents with a severe neurological phenotype carries both of these mutations in a compound heterozygous state. The p.Pro135Leu variant, however, is present in the proband’s mother and sibling as well, and is consistent with an autosomal dominant pattern linked to tonic-clonic and myoclonic epilepsy. In conclusion, we describe a single family in which TBC1D24 mutations cause expanded dominant and recessive phenotypes. In addition, we discuss and highlight that some variants in TBC1D24 might cause a dominant susceptibility to epilepsy

A de novo missense mutation in ZMYND11 is associated with global developmental delay, seizures and hypotonia

Recently, mutations in the zinc finger MYND-type containing 11 (ZMYND11) gene were identified in patients with autism spectrum disorders, intellectual disability, aggression, and complex neuropsychiatric features, supporting that this gene is implicated in 10p15.3 microdeletion syndrome. We report a novel de novo variant in the ZMYND11 gene (p.Ser421Asn) in a patient with a complex neurodevelopmental phenotype. The patient is a 24-yr-old Caucasian/Filipino female with seizures, global developmental delay, sensorineural hearing loss, hypotonia, dysmorphic features, and other features including a happy disposition and ataxic gait similar to Angelman syndrome. In addition, this patient had uncommon features including eosinophilic esophagitis and multiple, severe allergies not described in similar ZMYND11 cases. This new case further supports the association of ZMYND11 with autistic-like phenotypes and suggests that ZMYND11 should be included in the list of potentially causative candidate genes in cases with complex neurodevelopmental phenotypes.

Neonatal epileptic encephalopathy caused by de novo GNAO1 mutation misdiagnosed as atypical Rett syndrome: Cautions in interpretation of genomic test results

Epileptic encephalopathies are childhood brain disorders characterized by a variety of severe epilepsy syndromes that differ by the age of onset and seizure type. Until recently, the cause of many epileptic encephalopathies was unknown. Whole exome or whole genome sequencing has led to the identification of several causal genes in individuals with epileptic encephalopathy, and the list of genes has now expanded greatly. Genetic testing with epilepsy gene panels is now done quite early in the evaluation of children with epilepsy, following brain imaging, electroencephalogram, and metabolic profile. Early infantile epileptic encephalopathy (EIEE1; OMIM #308350) is the earliest of these age-dependent encephalopathies, manifesting as tonic spasms, myoclonic seizures, or partial seizures, with severely abnormal electroencephalogram, often showing a suppression-burst pattern. In this case study, we describe a 33-month-old female child with severe, neonatal onset epileptic encephalopathy. An infantile epilepsy gene panel test revealed 2 novel heterozygous variants in the MECP2 gene; a 70-bp deletion resulting in a frameshift and truncation (p.Lys377ProfsX9) thought to be pathogenic, and a 6-bp in-frame deletion (p.His371_372del), designated as a variant of unknown significance. Based on this test result, the diagnosis of atypical Rett syndrome (RTT) was made. Family-based targeted testing and segregation analysis, however, raised questions about the pathogenicity of these specific MECP2 variants. Whole exome sequencing was performed in this family trio, leading to the discovery of a rare, de novo, missense mutation in GNAO1 (p. Leu284Ser). De novo, heterozygous mutations in GNAO1 have been reported to cause early infantile epileptic encephalopathy-17 (EIEE17; OMIM 615473). The childs severe phenotype, the family history and segregation analysis of variants and prior reports of GNAO1-linked disease allowed us to conclude that the GNAO1 mutation, and not the MECP2 variants, was the cause of this childs neurological disease. With the increased use of genetic panels and whole exome sequencing, we will be confronted with lists of gene variants suspected to be pathogenic or of unknown significance. It is important to integrate clinical information, genetic testing that includes family members and correlates this with the published clinical and scientific literature, to help one arrive at the correct genetic diagnosis.

Hippocampal Transcriptomic Profiles: Subfield Vulnerability to Age and Cognitive Impairment

The current study employed next-generation RNA sequencing to examine gene expression differences related to brain aging, cognitive decline, and hippocampal subfields. Young and aged rats were trained on a spatial episodic memory task. Hippocampal regions CA1, CA3, and the dentate gyrus were isolated. Poly-A mRNA was examined using two different sequencing platforms, Illumina, and Ion Proton. The Illumina platform was used to generate seed lists of genes that were statistically differentially expressed across regions, ages, or in association with cognitive function. The gene lists were then retested using the data from the Ion Proton platform. The results indicate hippocampal subfield differences in gene expression and point to regional differences in vulnerability to aging. Aging was associated with increased expression of immune response-related genes, particularly in the dentate gyrus. For the memory task, impaired performance of aged animals was linked to the regulation of Ca2+ and synaptic function in region CA1. Finally, we provide a transcriptomic characterization of the three subfields regardless of age or cognitive status, highlighting and confirming a correspondence between cytoarchitectural boundaries and molecular profiling.

A Guide to Single-Cell Transcriptomics in Adult Rodent Brain: The Medium Spiny Neuron Transcriptome Revisited

Recent advances in single-cell technologies are paving the way to a comprehensive understanding of the cellular complexity in the brain. Protocols for single-cell transcriptomics combine a variety of sophisticated methods for the purpose of isolating the heavily interconnected and heterogeneous neuronal cell types in a relatively intact and healthy state. The emphasis of single-cell transcriptome studies has thus far been on comparing library generation and sequencing techniques that enable measurement of the minute amounts of starting material from a single cell. However, in order for data to be comparable, standardized cell isolation techniques are essential. Here, we analyzed and simplified methods for the different steps critically involved in single-cell isolation from brain. These include enzymatic digestion, tissue trituration, improved methods for efficient fluorescence-activated cell sorting in samples containing high degree of debris from the neuropil, and finally, highly region-specific cellular labeling compatible with use of stereotaxic coordinates. The methods are exemplified using medium spiny neurons (MSN) from dorsomedial striatum, a cell type that is clinically relevant for disorders of the basal ganglia, including psychiatric and neurodegenerative diseases. We present single-cell RNA sequencing (scRNA-Seq) data from D1 and D2 dopamine receptor expressing MSN subtypes. We illustrate the need for single-cell resolution by comparing to available population-based gene expression data of striatal MSN subtypes. Our findings contribute toward standardizing important steps of single-cell isolation from adult brain tissue to increase comparability of data. Furthermore, our data redefine the transcriptome of MSNs at unprecedented resolution by confirming established marker genes, resolving inconsistencies from previous gene expression studies, and identifying novel subtype-specific marker genes in this important cell type.

A novel FBXO28 frameshift mutation in a child with developmental delay, dysmorphic features, and intractable epilepsy: A second gene that may contribute to the 1q41-q42 deletion phenotype

Chromosome 1q41‐q42 deletions have recently been associated with a recognizable neurodevelopmental syndrome of early childhood (OMIM 612530). Within this group, a predominant phenotype of developmental delay (DD), intellectual disability (ID), epilepsy, distinct dysmorphology, and brain anomalies on magnetic resonance imaging/computed tomography has emerged. Previous reports of patients with de novo deletions at 1q41‐q42 have led to the identification of an evolving smallest region of overlap which has included several potentially causal genes including DISP1, TP53BP2, and FBXO28. In a recent report, a cohort of patients with de novo mutations in WDR26 was described that shared many of the clinical features originally described in the 1q41‐q42 microdeletion syndrome (MDS). Here, we describe a novel germline FBXO28 frameshift mutation in a 3‐year‐old girl with intractable epilepsy, ID, DD, and other features which overlap those of the 1q41‐q42 MDS. Through a familial whole‐exome sequencing study, we identified a de novo FBXO28 c.972_973delACinsG (p.Arg325GlufsX3) frameshift mutation in the proband. The frameshift and resulting premature nonsense mutation have not been reported in any genomic database. This child does not have a large 1q41‐q42 deletion, nor does she harbor a WDR26 mutation. Our case joins a previously reported patient also in whom FBXO28 was affected but WDR26 was not. These findings support the idea that FBXO28 is a monogenic disease gene and contributes to the complex neurodevelopmental phenotype of the 1q41‐q42 gene deletion syndrome.