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1.  Gene-ontology enrichment analysis in two independent family-based samples highlights biologically plausible processes for autism spectrum disorders 
European Journal of Human Genetics  2011;19(10):1082-1089.
Recent genome-wide association studies (GWAS) have implicated a range of genes from discrete biological pathways in the aetiology of autism. However, despite the strong influence of genetic factors, association studies have yet to identify statistically robust, replicated major effect genes or SNPs. We apply the principle of the SNP ratio test methodology described by O'Dushlaine et al to over 2100 families from the Autism Genome Project (AGP). Using a two-stage design we examine association enrichment in 5955 unique gene-ontology classifications across four groupings based on two phenotypic and two ancestral classifications. Based on estimates from simulation we identify excess of association enrichment across all analyses. We observe enrichment in association for sets of genes involved in diverse biological processes, including pyruvate metabolism, transcription factor activation, cell-signalling and cell-cycle regulation. Both genes and processes that show enrichment have previously been examined in autistic disorders and offer biologically plausibility to these findings.
PMCID: PMC3190264  PMID: 21522181
autism; genome-wide association analysis; pathway analysis; family-based association test; gene ontology
2.  A critical role for human caspase-4 in endotoxin sensitivity 
Response to endotoxins is an important part of the organismal reaction to gram-negative bacteria, and plays a critical role in sepsis and septic shock, as well as other conditions such as metabolic endotoxemia. Humans are generally more sensitive to endotoxins when compared to experimental animals such as mouse. Inflammatory caspases mediate endotoxin-induced interelukin-1β (IL-1β) secretion and lethality in mouse, and caspase-4 is an inflammatory caspase that is found in the human, and not mouse, genome. To test whether caspase-4 is involved in endotoxin sensitivity, we developed a transgenic mouse expressing human caspase-4 in its genomic context. Caspase-4 transgenic mice exhibited significantly higher endotoxin sensitivity, as measured by enhanced cytokine secretion and lethality following lipopolysaccharide (LPS) challenge. Using bone marrow-derived macrophages (BMDMs), we then observed that caspase-4 can support activation of caspase-1 and secretion of IL-1β and IL-18 in response to priming signals (LPS or Pam3CSK4) alone, without the need for second signals to stimulate the assembly of the inflammasome. These findings indicate that the regulation of caspase-1 activity by human caspase-4 could represent unique mechanism in humans, as compared to laboratory rodents, and may partially explain the higher sensitivity to endotoxins observed in humans. Regulation of the expression, activation, or activity of caspase-4 therefore represent therapeutic targets for systemic inflammatory response syndrome (SIRS), sepsis, septic shock, and related disorders.
PMCID: PMC4066208  PMID: 24879791
3.  Slc25a12 disruption alters myelination and neurofilaments: A model for a hypomyelination syndrome and childhood neurodevelopmental disorders 
Biological psychiatry  2009;67(9):887-894.
SLC25A12, a susceptibility gene for autism spectrum disorders (ASDs) that is mutated in a neurodevelopmental syndrome, encodes a mitochondrial aspartate/glutamate carrier (AGC1). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and ATP production.
We characterized mice with a disruption of the Slc25a12 gene, followed by confirmatory in vitro studies.
Slc25a12-knockout mice, which showed no AGC1 by immunoblotting, were born normally but displayed delayed development and died around 3 weeks after birth. In P13-14 knockout brains, the brains were smaller with no obvious alteration in gross structure. However, we found a reduction in myelin basic protein (MBP)-positive fibers, consistent with a previous report. Furthermore, the neocortex of knockout mice contained abnormal neurofilamentous accumulations in neurons, suggesting defective axonal transport and/or neurodegeneration. Slice cultures prepared from knockout mice also showed a myelination defect, and reduction of Slc25a12 in rat primary oligodendrocytes led to a cellautonomous reduction in MBP expression. Myelin deficits in slice cultures from knockout mice could be reversed by administration of pyruvate, indicating that reduction in AGC1 activity leads to reduced production of aspartate/N-acetyl aspartate (NAA) and/or alterations in the NADH/NAD+ ratio, resulting in myelin defects.
Our data implicate AGC1 activity in myelination and in neuronal structure, and indicate that while loss of AGC1 leads to hypomyelination and neuronal changes, subtle alterations in AGC1 expression could affect brain development contributing to increased autism susceptibility.
PMCID: PMC4067545  PMID: 20015484
Malate/aspartate shuttle; mitochondria; N-acetyl aspartate (NAA); neuron-oligodendrocyte interactions; pyruvate
4.  A Critical Role for Human Caspase-4 in Endotoxin Sensitivity 
Response to endotoxins is an important part of the organismal reaction to Gram-negative bacteria and plays a critical role in sepsis and septic shock, as well as other conditions such as metabolic endotoxemia. Humans are generally more sensitive to endotoxins when compared with experimental animals such as mice. Inflammatory caspases mediate endotoxin-induced IL-1β secretion and lethality in mice, and caspase-4 is an inflammatory caspase that is found in the human, and not mouse, genome. To test whether caspase-4 is involved in endotoxin sensitivity, we developed a transgenic mouse expressing human caspase-4 in its genomic context. Caspase-4 transgenic mice exhibited significantly higher endotoxin sensitivity, as measured by enhanced cytokine secretion and lethality following LPS challenge. Using bone marrow–derived macrophages, we then observed that caspase-4 can support activation of caspase-1 and secretion of IL-1β and IL-18 in response to priming signals (LPS or Pam3CSK4) alone, without the need for second signals to stimulate the assembly of the inflammasome. These findings indicate that the regulation of caspase-1 activity by human caspase-4 could represent a unique mechanism in humans, as compared with laboratory rodents, and may partially explain the higher sensitivity to endotoxins observed in humans. Regulation of the expression, activation, or activity of caspase-4 therefore represents targets for systemic inflammatory response syndrome, sepsis, septic shock, and related disorders.
PMCID: PMC4066208  PMID: 24879791
5.  Ultrastructural analyses in the hippocampus CA1 field in Shank3-deficient mice 
Molecular Autism  2015;6:41.
The genetics of autism spectrum disorder (hereafter referred to as “autism”) are rapidly unfolding, with a significant increase in the identification of genes implicated in the disorder. Many of these genes are part of a complex landscape of genetic variants that are thought to act together to cause the behavioral phenotype associated with autism. One of the few single-locus causes of autism involves a mutation in the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene. Previous electrophysiological studies in mice with Shank3 mutations demonstrated impairment in synaptic long-term potentiation, suggesting a potential disruption at the synapse.
To understand how variants in SHANK3 would lead to such impairments and manifest in the brain of patients with autism, we assessed the presence of synaptic pathology in Shank3-deficient mice at 5 weeks and 3 months of age, focusing on the stratum radiatum of the CA1 field. This study analyzed both Shank3 heterozygous and homozygous mice using an electron microscopy approach to determine whether there is a morphological correlate to the synaptic functional impairment.
As both synaptic strength and plasticity are affected in Shank3-deficient mice, we hypothesized that there would be a reduction in synapse density, postsynaptic density length, and perforated synapse density. No differences were found in most parameters assessed. However, Shank3 heterozygotes had significantly higher numbers of perforated synapses at 5 weeks compared to 3 months of age and significantly higher numbers of perforated synapses compared to 5-week-old wildtype and Shank3 homozygous mice.
Although this finding represents preliminary evidence for ultrastructural alterations, it suggests that while major structural changes seem to be compensated for in Shank3-deficient mice, more subtle morphological alterations, affecting synaptic structure, may take place in an age-dependent manner.
PMCID: PMC4486760  PMID: 26137200
Autism; Shank3; Electron microscopy; CA1; Hippocampus; Stratum radiatum; Synapse
7.  Synaptic, transcriptional, and chromatin genes disrupted in autism 
De Rubeis, Silvia | He, Xin | Goldberg, Arthur P. | Poultney, Christopher S. | Samocha, Kaitlin | Cicek, A Ercument | Kou, Yan | Liu, Li | Fromer, Menachem | Walker, Susan | Singh, Tarjinder | Klei, Lambertus | Kosmicki, Jack | Fu, Shih-Chen | Aleksic, Branko | Biscaldi, Monica | Bolton, Patrick F. | Brownfeld, Jessica M. | Cai, Jinlu | Campbell, Nicholas J. | Carracedo, Angel | Chahrour, Maria H. | Chiocchetti, Andreas G. | Coon, Hilary | Crawford, Emily L. | Crooks, Lucy | Curran, Sarah R. | Dawson, Geraldine | Duketis, Eftichia | Fernandez, Bridget A. | Gallagher, Louise | Geller, Evan | Guter, Stephen J. | Hill, R. Sean | Ionita-Laza, Iuliana | Gonzalez, Patricia Jimenez | Kilpinen, Helena | Klauck, Sabine M. | Kolevzon, Alexander | Lee, Irene | Lei, Jing | Lehtimäki, Terho | Lin, Chiao-Feng | Ma'ayan, Avi | Marshall, Christian R. | McInnes, Alison L. | Neale, Benjamin | Owen, Michael J. | Ozaki, Norio | Parellada, Mara | Parr, Jeremy R. | Purcell, Shaun | Puura, Kaija | Rajagopalan, Deepthi | Rehnström, Karola | Reichenberg, Abraham | Sabo, Aniko | Sachse, Michael | Sanders, Stephan J. | Schafer, Chad | Schulte-Rüther, Martin | Skuse, David | Stevens, Christine | Szatmari, Peter | Tammimies, Kristiina | Valladares, Otto | Voran, Annette | Wang, Li-San | Weiss, Lauren A. | Willsey, A. Jeremy | Yu, Timothy W. | Yuen, Ryan K.C. | Cook, Edwin H. | Freitag, Christine M. | Gill, Michael | Hultman, Christina M. | Lehner, Thomas | Palotie, Aarno | Schellenberg, Gerard D. | Sklar, Pamela | State, Matthew W. | Sutcliffe, James S. | Walsh, Christopher A. | Scherer, Stephen W. | Zwick, Michael E. | Barrett, Jeffrey C. | Cutler, David J. | Roeder, Kathryn | Devlin, Bernie | Daly, Mark J. | Buxbaum, Joseph D.
Nature  2014;515(7526):209-215.
The genetic architecture of autism spectrum disorder involves the interplay of common and rare variation and their impact on hundreds of genes. Using exome sequencing, analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, and a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic, transcriptional, and chromatin remodeling pathways. These include voltage-gated ion channels regulating propagation of action potentials, pacemaking, and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodelers, prominently histone post-translational modifications involving lysine methylation/demethylation.
PMCID: PMC4402723  PMID: 25363760
8.  Understanding autism in the light of sex/gender 
Molecular Autism  2015;6:24.
PMCID: PMC4429357  PMID: 25973161
9.  Phenotypic and functional analysis of SHANK3 stop mutations identified in individuals with ASD and/or ID 
Molecular Autism  2015;6:23.
SHANK proteins are crucial for the formation and plasticity of excitatory synapses. Although mutations in all three SHANK genes are associated with autism spectrum disorder (ASD), SHANK3 appears to be the major ASD gene with a prevalence of approximately 0.5% for SHANK3 mutations in ASD, with higher rates in individuals with ASD and intellectual disability (ID). Interestingly, the most relevant mutations are typically de novo and often are frameshift or nonsense mutations resulting in a premature stop and a truncation of SHANK3 protein.
We analyzed three different SHANK3 stop mutations that we identified in individuals with ASD and/or ID, one novel (c.5008A > T) and two that we recently described (c.1527G > A, c.2497delG). The mutations were inserted into the human SHANK3a sequence and analyzed for effects on subcellular localization and neuronal morphology when overexpressed in rat primary hippocampal neurons.
Clinically, all three individuals harboring these mutations had global developmental delays and ID. In our in vitro assay, c.1527G > A and c.2497delG both result in proteins that lack most of the SHANK3a C-terminus and accumulate in the nucleus of transfected cells. Cells expressing these mutants exhibit converging morphological phenotypes including reduced complexity of the dendritic tree, less spines, and less excitatory, but not inhibitory synapses. In contrast, the truncated protein based on c.5008A > T, which lacks only a short part of the sterile alpha motif (SAM) domain in the very SHANK3a C-terminus, does not accumulate in the nucleus and has minor effects on neuronal morphology.
In spite of the prevalence of SHANK3 disruptions in ASD and ID, only a few human mutations have been functionally characterized; here we characterize three additional mutations. Considering the transcriptional and functional complexity of SHANK3 in healthy neurons, we propose that any heterozygous stop mutation in SHANK3 will lead to a dysequilibrium of SHANK3 isoform expression and alterations in the stoichiometry of SHANK3 protein complexes, resulting in a distinct perturbation of neuronal morphology. This could explain why the clinical phenotype in all three individuals included in this study remains quite severe - regardless of whether there are disruptions in one or more SHANK3 interaction domains.
Electronic supplementary material
The online version of this article (doi:10.1186/s13229-015-0020-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4455919  PMID: 26045941
ASD; Autism; SHANK3; Intellectual disability; Nucleus; Dendrite; Spine; Synapse
10.  Network- and Attribute-Based Classifiers Can Prioritize Genes and Pathways for Autism Spectrum Disorders and for Intellectual Disability 
Autism spectrum disorders (ASD) are a group of related neurodevelopmental disorders with significant combined prevalence (~1%) and high heritability. Dozens of individually rare genes and loci associated with high-risk for ASD have been identified, which overlap extensively with genes for intellectual disability (ID). However, studies indicate that there may be hundreds of genes that remain to be identified. The advent of inexpensive massively parallel nucleotide sequencing can reveal the genetic underpinnings of heritable complex diseases, including ASD and ID. However, whole exome sequencing (WES) and whole genome sequencing (WGS) provides an embarrassment of riches, where many candidate variants emerge. It has been argued that genetic variation for ASD and ID will cluster in genes involved in distinct pathways and protein complexes. For this reason, computational methods that prioritize candidate genes based on additional functional information such as protein-protein interactions or association with specific canonical or empirical pathways, or other attributes, can be useful. In this study we applied several supervised learning approaches to prioritize ASD or ID disease gene candidates based on curated lists of known ASD and ID disease genes. We implemented two network-based classifiers and one attribute-based classifier to show that we can rank and classify known, and predict new, genes for these neurodevelopmental disorders. We also show that ID and ASD share common pathways that perturb an overlapping synaptic regulatory subnetwork. We also show that features relating to neuronal phenotypes in mouse knockouts can help in classifying neurodevelopmental genes. Our methods can be applied broadly to other diseases helping in prioritizing newly identified genetic variation that emerge from disease gene discovery based on WES and WGS.
PMCID: PMC3505691  PMID: 22499558
High-throughput sequencing; massively parallel sequencing; gene discovery; networks; pathways; neurodevelopmental disorders; classifiers; support vector machine
11.  Linking oligodendrocyte and myelin dysfunction to neurocircuitry abnormalities in schizophrenia 
Progress in neurobiology  2010;93(1):13-24.
Multiple lines of evidence in schizophrenia, from brain imaging, studies in postmortem brains, and genetic association studies, have implicated oligodendrocyte and myelin dysfunction in this disease. Recent studies suggest that oligodendrocyte and myelin dysfunction leads to changes in synaptic formation and function, which could lead to cognitive dysfunction, a core symptom of schizophrenia. Furthermore, there is accumulating data linking oligodendrocyte and myelin dysfunction with dopamine and glutamate abnormalities, both of which are found in schizophrenia. These findings implicate oligodendrocyte and myelin dysfunction as a primary change in schizophrenia, not only as secondary consequences of the illness or treatment. Strategies targeting oligodendrocyte and myelin abnormalities could therefore provide therapeutic opportunities for patients suffering from schizophrenia.
PMCID: PMC3622281  PMID: 20950668
myelin; gene expression; genetic association; brain imaging; oligodendrocyte; synaptic plasticity; dopamine; glutamate
12.  A framework for the interpretation of de novo mutation in human disease 
Nature genetics  2014;46(9):944-950.
Spontaneously arising (‘de novo’) mutations play an important role in medical genetics. For diseases with extensive locus heterogeneity – such as autism spectrum disorders (ASDs) – the signal from de novo mutations (DNMs) is distributed across many genes, making it difficult to distinguish disease-relevant mutations from background variation. We provide a statistical framework for the analysis of DNM excesses per gene and gene set by calibrating a model of de novo mutation. We applied this framework to DNMs collected from 1,078 ASD trios and – while affirming a significant role for loss-of-function (LoF) mutations – found no excess of de novo LoF mutations in cases with IQ above 100, suggesting that the role of DNMs in ASD may reside in fundamental neurodevelopmental processes. We also used our model to identify ~1,000 genes that are significantly lacking functional coding variation in non-ASD samples and are enriched for de novo LoF mutations identified in ASD cases.
PMCID: PMC4222185  PMID: 25086666
13.  Age-at-Onset in Late Onset Alzheimer Disease is Modified by Multiple Genetic Loci 
JAMA neurology  2014;71(11):1394-1404.
As APOE locus variants contribute to both risk of late-onset Alzheimer disease and differences in age-at-onset, it is important to know if other established late-onset Alzheimer disease risk loci also affect age-at-onset in cases.
To investigate the effects of known Alzheimer disease risk loci in modifying age-at-onset, and to estimate their cumulative effect on age-at-onset variation, using data from genome-wide association studies in the Alzheimer’s Disease Genetics Consortium (ADGC).
Design, Setting and Participants
The ADGC comprises 14 case-control, prospective, and family-based datasets with data on 9,162 Caucasian participants with Alzheimer’s occurring after age 60 who also had complete age-at-onset information, gathered between 1989 and 2011 at multiple sites by participating studies. Data on genotyped or imputed single nucleotide polymorphisms (SNPs) most significantly associated with risk at ten confirmed LOAD loci were examined in linear modeling of AAO, and individual dataset results were combined using a random effects, inverse variance-weighted meta-analysis approach to determine if they contribute to variation in age-at-onset. Aggregate effects of all risk loci on AAO were examined in a burden analysis using genotype scores weighted by risk effect sizes.
Main Outcomes and Measures
Age at disease onset abstracted from medical records among participants with late-onset Alzheimer disease diagnosed per standard criteria.
Analysis confirmed association of APOE with age-at-onset (rs6857, P=3.30×10−96), with associations in CR1 (rs6701713, P=7.17×10−4), BIN1 (rs7561528, P=4.78×10−4), and PICALM (rs561655, P=2.23×10−3) reaching statistical significance (P<0.005). Risk alleles individually reduced age-at-onset by 3-6 months. Burden analyses demonstrated that APOE contributes to 3.9% of variation in age-at-onset (R2=0.220) over baseline (R2=0.189) whereas the other nine loci together contribute to 1.1% of variation (R2=0.198).
Conclusions and Relevance
We confirmed association of APOE variants with age-at-onset among late-onset Alzheimer disease cases and observed novel associations with age-at-onset in CR1, BIN1, and PICALM. In contrast to earlier hypothetical modeling, we show that the combined effects of Alzheimer disease risk variants on age-at-onset are on the scale of, but do not exceed, the APOE effect. While the aggregate effects of risk loci on age-at-onset may be significant, additional genetic contributions to age-at-onset are individually likely to be small.
PMCID: PMC4314944  PMID: 25199842
Alzheimer Disease; Alzheimer Disease Genetics; Alzheimer’s Disease - Pathophysiology; Genetics of Alzheimer Disease; Aging
14.  Most genetic risk for autism resides with common variation 
Nature genetics  2014;46(8):881-885.
A key component of genetic architecture is the allelic spectrum influencing trait variability. For autism spectrum disorder (henceforth autism) the nature of its allelic spectrum is uncertain. Individual risk genes have been identified from rare variation, especially de novo mutations1–8. From this evidence one might conclude that rare variation dominates its allelic spectrum, yet recent studies show that common variation, individually of small effect, has substantial impact en masse9,10. At issue is how much of an impact relative to rare variation. Using a unique epidemiological sample from Sweden, novel methods that distinguish total narrow-sense heritability from that due to common variation, and by synthesizing results from other studies, we reach several conclusions about autism’s genetic architecture: its narrow-sense heritability is ≈54% and most traces to common variation; rare de novo mutations contribute substantially to individuals’ liability; still their contribution to variance in liability, 2.6%, is modest compared to heritable variation.
PMCID: PMC4137411  PMID: 25038753
15.  Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder 
Nature genetics  2011;44(1):78-84.
Attention deficit hyperactivity disorder (ADHD) is a common, heritable neuropsychiatric disorder of unknown etiology. We performed a whole-genome copy number variation (CNV) study on 1,013 cases with ADHD and 4,105 healthy children of European ancestry using 550,000 SNPs. We evaluated statistically significant findings in multiple independent cohorts, with a total of 2,493 cases with ADHD and 9,222 controls of European ancestry, using matched platforms. CNVs affecting metabotropic glutamate receptor genes were enriched across all cohorts (P = 2.1 × 10−9). We saw GRM5 (encoding glutamate receptor, metabotropic 5) deletions in ten cases and one control (P = 1.36 × 10−6). We saw GRM7 deletions in six cases, and we saw GRM8 deletions in eight cases and no controls. GRM1 was duplicated in eight cases. We experimentally validated the observed variants using quantitative RT-PCR. A gene network analysis showed that genes interacting with the genes in the GRM family are enriched for CNVs in ~10% of the cases (P = 4.38 × 10−10) after correction for occurrence in the controls. We identified rare recurrent CNVs affecting glutamatergic neurotransmission genes that were overrepresented in multiple ADHD cohorts.
PMCID: PMC4310555  PMID: 22138692
16.  A pilot controlled trial of insulin-like growth factor-1 in children with Phelan-McDermid syndrome 
Molecular Autism  2014;5:54.
Autism spectrum disorder (ASD) is now understood to have multiple genetic risk genes and one example is SHANK3. SHANK3 deletions and mutations disrupt synaptic function and result in Phelan-McDermid syndrome (PMS), which causes a monogenic form of ASD with a frequency of at least 0.5% of ASD cases. Recent evidence from preclinical studies with mouse and human neuronal models of SHANK3 deficiency suggest that insulin-like growth factor-1 (IGF-1) can reverse synaptic plasticity and motor learning deficits. The objective of this study was to pilot IGF-1 treatment in children with PMS to evaluate safety, tolerability, and efficacy for core deficits of ASD, including social impairment and restricted and repetitive behaviors.
Nine children with PMS aged 5 to 15 were enrolled in a placebo-controlled, double-blind, crossover design study, with 3 months of treatment with IGF-1 and 3 months of placebo in random order, separated by a 4-week wash-out period.
Compared to the placebo phase, the IGF-1 phase was associated with significant improvement in both social impairment and restrictive behaviors, as measured by the Aberrant Behavior Checklist and the Repetitive Behavior Scale, respectively. IGF-1 was found to be well tolerated and there were no serious adverse events in any participants.
This study establishes the feasibility of IGF-1 treatment in PMS and contributes pilot data from the first controlled treatment trial in the syndrome. Results also provide proof of concept to advance knowledge about developing targeted treatments for additional causes of ASD associated with impaired synaptic development and function.
PMCID: PMC4326443  PMID: 25685306
17.  Identification of Rare Causal Variants in Sequence-Based Studies: Methods and Applications to VPS13B, a Gene Involved in Cohen Syndrome and Autism 
PLoS Genetics  2014;10(12):e1004729.
Pinpointing the small number of causal variants among the abundant naturally occurring genetic variation is a difficult challenge, but a crucial one for understanding precise molecular mechanisms of disease and follow-up functional studies. We propose and investigate two complementary statistical approaches for identification of rare causal variants in sequencing studies: a backward elimination procedure based on groupwise association tests, and a hierarchical approach that can integrate sequencing data with diverse functional and evolutionary conservation annotations for individual variants. Using simulations, we show that incorporation of multiple bioinformatic predictors of deleteriousness, such as PolyPhen-2, SIFT and GERP++ scores, can improve the power to discover truly causal variants. As proof of principle, we apply the proposed methods to VPS13B, a gene mutated in the rare neurodevelopmental disorder called Cohen syndrome, and recently reported with recessive variants in autism. We identify a small set of promising candidates for causal variants, including two loss-of-function variants and a rare, homozygous probably-damaging variant that could contribute to autism risk.
Author Summary
Sequencing technologies allow identification of genetic variants down to single base resolution for a whole human genome. The vast majority of these variants (over 90%) are rare, with population frequencies less than 1%. Furthermore, in a specific study, many of the variants identified are not associated with the disease of interest, and identification of the small proportion of truly causal variants is a difficult task. Clearly, for causal variants that are rare enough to only appear a few times in a study, observed frequencies in cases and controls are not enough to distinguish them from the vast majority of random variation, and rich functional annotations can help identify the causal variants. Here we propose to develop a set of statistical methods that leverage diverse functional genomics annotations with sequencing data to identify a small set of potentially causal variants and estimate their effects. Pinpointing a subset of potentially causal variants is crucial for understanding precise biological mechanisms, and for further experimental functional studies.
PMCID: PMC4263785  PMID: 25502226
18.  The ACMSD gene, involved in tryptophan metabolism, is mutated in a family with cortical myoclonus, epilepsy, and parkinsonism 
Journal of molecular medicine (Berlin, Germany)  2013;91(12):10.1007/s00109-013-1075-4.
Familial cortical myoclonic tremor and epilepsy is a phenotypically and genetically heterogeneous autosomal dominant disorder characterized by the presence of cortical myoclonic tremor and epilepsy that is often accompanied of additional neurological features. Despite the numerous familial studies performed and the number of loci identified, there is no gene associated with this syndrome. It is expected that through the application of novel genomic technologies, such as whole exome sequencing and whole genome sequencing, a substantial number of novel genes will come to light in the coming years. In this study, we describe the identification of two disease-segregating mutations in a large family featuring cortical myoclonic tremor with epilepsy and parkinsonism. Due to the previous association of ACMSD deficiency with the development of epileptic seizures, we concluded that the identified nonsense mutation in the ACMSD gene, which encodes for a critical enzyme of the kynurenine pathway of the tryptophan metabolism, is the disease-segregating mutation most likely to be responsible for the phenotype described in our family. This finding not only reveals the identification of the first gene associated with familial cortical myoclonic tremor and epilepsy but also discloses the kynurenine pathway as a potential therapeutic target for the treatment of this devastating syndrome.
PMCID: PMC3833949  PMID: 23955123
FCMTE; Whole Exome Sequencing; ACMSD; Kynurenine Pathway
19.  Family-based association tests for sequence data, and comparisons with population-based association tests 
European Journal of Human Genetics  2013;21(10):1158-1162.
Recent advances in high-throughput sequencing technologies make it increasingly more efficient to sequence large cohorts for many complex traits. We discuss here a class of sequence-based association tests for family-based designs that corresponds naturally to previously proposed population-based tests, including the classical Burden and variance-component tests. This framework allows for a direct comparison between the powers of sequence-based association tests with family- vs population-based designs. We show that for dichotomous traits using family-based controls results in similar power levels as the population-based design (although at an increased sequencing cost for the family-based design), while for continuous traits (in random samples, no ascertainment) the population-based design can be substantially more powerful. A possible disadvantage of population-based designs is that they can lead to increased false-positive rates in the presence of population stratification, while the family-based designs are robust to population stratification. We show also an application to a small exome-sequencing family-based study on autism spectrum disorders. The tests are implemented in publicly available software.
PMCID: PMC3778346  PMID: 23386037
family- and population-based association tests; sequence data; burden and variance-component tests
20.  Whole exome sequencing reveals minimal differences between cell line and whole blood derived DNA 
Genomics  2013;102(4):10.1016/j.ygeno.2013.05.005.
Two common sources of DNA for whole exome sequencing (WES) are whole blood (WB) and immortalized lymphoblastoid cell line (LCL). However, it is possible that LCLs have a substantially higher rate of mutation than WB, causing concern for their use in sequencing studies. We compared results from paired WB and LCL DNA samples for 16 subjects, using LCLs of low passage number (<5). Using a standard analysis pipeline we detected a large number of discordant genotype calls (approximately 50 per subject) that we segregated into categories of “confidence” based on read-level quality metrics. From these categories and validation by Sanger sequencing, we estimate that the vast majority of the candidate differences were false positives and that our categories were effective in predicting valid sequence differences, including LCLs with putative mosaicism for the non-reference allele (3–4 per exome). These results validate the use of DNA from LCLs of low passage number for exome sequencing.
PMCID: PMC3812417  PMID: 23743231
graphical diagnostics; lymphoblastoid cell line; mosaicism; sequence variant call; strand bias; somatic mutation
21.  Genome-Wide Association Meta-analysis of Neuropathologic Features of Alzheimer's Disease and Related Dementias 
PLoS Genetics  2014;10(9):e1004606.
Alzheimer's disease (AD) and related dementias are a major public health challenge and present a therapeutic imperative for which we need additional insight into molecular pathogenesis. We performed a genome-wide association study and analysis of known genetic risk loci for AD dementia using neuropathologic data from 4,914 brain autopsies. Neuropathologic data were used to define clinico-pathologic AD dementia or controls, assess core neuropathologic features of AD (neuritic plaques, NPs; neurofibrillary tangles, NFTs), and evaluate commonly co-morbid neuropathologic changes: cerebral amyloid angiopathy (CAA), Lewy body disease (LBD), hippocampal sclerosis of the elderly (HS), and vascular brain injury (VBI). Genome-wide significance was observed for clinico-pathologic AD dementia, NPs, NFTs, CAA, and LBD with a number of variants in and around the apolipoprotein E gene (APOE). GalNAc transferase 7 (GALNT7), ATP-Binding Cassette, Sub-Family G (WHITE), Member 1 (ABCG1), and an intergenic region on chromosome 9 were associated with NP score; and Potassium Large Conductance Calcium-Activated Channel, Subfamily M, Beta Member 2 (KCNMB2) was strongly associated with HS. Twelve of the 21 non-APOE genetic risk loci for clinically-defined AD dementia were confirmed in our clinico-pathologic sample: CR1, BIN1, CLU, MS4A6A, PICALM, ABCA7, CD33, PTK2B, SORL1, MEF2C, ZCWPW1, and CASS4 with 9 of these 12 loci showing larger odds ratio in the clinico-pathologic sample. Correlation of effect sizes for risk of AD dementia with effect size for NFTs or NPs showed positive correlation, while those for risk of VBI showed a moderate negative correlation. The other co-morbid neuropathologic features showed only nominal association with the known AD loci. Our results discovered new genetic associations with specific neuropathologic features and aligned known genetic risk for AD dementia with specific neuropathologic changes in the largest brain autopsy study of AD and related dementias.
Author Summary
Alzheimer's disease (AD) and related dementias are a major public health challenge and present a therapeutic imperative for which we need additional insight into molecular pathogenesis. We performed a genome-wide association study (GWAS), as well as an analysis of known genetic risk loci for AD dementia, using data from 4,914 brain autopsies. Genome-wide significance was observed for 7 genes and pathologic features of AD and related diseases. Twelve of the 22 genetic risk loci for clinically-defined AD dementia were confirmed in our pathologic sample. Correlation of effect sizes for risk of AD dementia with effect size for hallmark pathologic features of AD were strongly positive and linear. Our study discovered new genetic associations with specific pathologic features and aligned known genetic risk for AD dementia with specific pathologic changes in a large brain autopsy study of AD and related dementias.
PMCID: PMC4154667  PMID: 25188341
22.  The Sac1 domain of SYNJ1 identified mutated in a family with early-onset progressive parkinsonism with generalized seizures 
Human mutation  2013;34(9):1200-1207.
This study aimed to elucidate the genetic causes underlying early-onset parkinsonism (EOP) in a consanguineous Iranian family. To attain this, homozygosity mapping and whole-exome sequencing were performed. As a result, a homozygous mutation (c.773G>A; p.Arg258Gln) lying within the NH2-terminal Sac1-like inositol phosphatase domain of polyphosphoinositide phosphatase synaptojanin 1 (SYNJ1), which has been implicated in the regulation of endocytic traffic at synapses, was identified as the disease-segregating mutation. This mutation impaired the phosphatase activity SYNJ1 against its Sac1 domain substrates in vitro. We concluded that the SYNJ1 mutation identified here is responsible for the EOP phenotype seen in our patients probably due to deficiencies in its phosphatase activity and consequent impairment of its synaptic functions. Our finding not only opens new avenues of investigation in the synaptic dysfunction mechanisms associated with parkinsonism, but also suggests phosphoinositide metabolism as a novel therapeutic target for parkinsonism.
PMCID: PMC3790461  PMID: 23804563
Homozygosity mapping; whole exome sequencing; SYNJ1; autosomal recessive parkinsonism
23.  Introduction of the human AVPR1A gene substantially alters brain receptor expression patterns and enhances aspects of social behavior in transgenic mice 
Disease Models & Mechanisms  2014;7(8):1013-1022.
Central arginine vasopressin receptor 1A (AVPR1A) modulates a wide range of behaviors, including stress management and territorial aggression, as well as social bonding and recognition. Inter- and intra-species variations in the expression pattern of AVPR1A in the brain and downstream differential behavioral phenotypes have been attributed to differences in the non-coding regions of the AVPR1A gene, including polymorphic elements within upstream regulatory areas. Gene association studies have suggested a link between AVPR1A polymorphisms and autism, and AVPR1A has emerged as a potential pharmacological target for treatment of social cognitive impairments and mood and anxiety disorders. To further investigate the genetic mechanism giving rise to species differences in AVPR1A expression patterns and associated social behaviors, and to create a preclinical mouse model useful for screening drugs targeting AVPR1A, we engineered and extensively characterized bacterial artificial chromosome (BAC) transgenic mice harboring the entire human AVPR1A locus with the surrounding regulatory elements. Compared with wild-type animals, the humanized mice displayed a more widely distributed ligand-AVPR1A binding pattern, which overlapped with that of primates. Furthermore, humanized AVPR1A mice displayed increased reciprocal social interactions compared with wild-type animals, but no differences in social approach and preference for social novelty were observed. Aspects of learning and memory, specifically novel object recognition and spatial relocation recognition, were unaffected. The biological alterations in humanized AVPR1A mice resulted in the rescue of the prepulse inhibition impairments that were observed in knockout mice, indicating conserved functionality. Although further behavioral paradigms and additional cohorts need to be examined in humanized AVPR1A mice, the results demonstrate that species-specific variations in the genomic content of regulatory regions surrounding the AVPR1A locus are responsible for differential receptor protein expression patterns across species and that they are likely to contribute to species-specific behavioral variation. The humanized AVPR1A mouse is a potential preclinical model for further understanding the regulation of receptor gene expression and the impact of variation in receptor expression on behaviors, and should be useful for screening drugs targeting human AVPR1A, taking advantage of the expression of human AVPR1A in human-relevant brain regions.
PMCID: PMC4107330  PMID: 24924430
AVPR1A; Humanized mouse; Social behavior; Species-specific; Microsatellite; Autism
24.  Regeneration in the Era of Functional Genomics and Gene Network Analysis 
The Biological bulletin  2011;221(1):18-34.
What gives an organism the ability to regrow tissues and to recover function where another organism fails is the central problem of regenerative biology. The challenge is to describe the mechanisms of regeneration at the molecular level, delivering detailed insights into the many components that are cross-regulated. In other words, a broad, yet deep dissection of the system-wide network of molecular interactions is needed. Functional genomics has been used to elucidate gene regulatory networks (GRNs) in developing tissues, which, like regeneration, are complex systems. Therefore, we reason that the GRN approach, aided by next generation technologies, can also be applied to study the molecular mechanisms underlying the complex functions of regeneration. We ask what characteristics a model system must have to support a GRN analysis. Our discussion focuses on regeneration in the central nervous system, where loss of function has particularly devastating consequences for an organism. We examine a cohort of cells conserved across all vertebrates, the reticulospinal (RS) neurons, which lend themselves well to experimental manipulations. In the lamprey, a jawless vertebrate, there are giant RS neurons whose large size and ability to regenerate make them particularly suited for a GRN analysis. Adding to their value, a distinct subset of lamprey RS neurons reproducibly fail to regenerate, presenting an opportunity for side-by-side comparison of gene networks that promote or inhibit regeneration. Thus, determining the GRN for regeneration in RS neurons will provide a mechanistic understanding of the fundamental cues that lead to success or failure to regenerate.
PMCID: PMC4109899  PMID: 21876108
25.  Evidence against a role for rare ADAM10 mutations in sporadic Alzheimer Disease 
Neurobiology of aging  2010;33(2):416-417.e3.
The Alzheimer amyloid protein precursor (APP) is subject to proteolysis by ADAM10 and ADAM17, precluding the formation of Aβ. Recently, coding variations in ADAM10 resulting in altered function have been reported in familial Alzheimer disease (AD). We carried out a large-scale (n=576: Controls, 271; AD, 305) resequencing study of ADAM10 in sporadic AD. Our results do not support a significant role for ADAM10 mutations in AD. Our results also make it clear that the careful examination of ancestry required in any case-control comparison is especially true with rare variations, where even a very small number of variations might form the basis of scientific conclusions.
PMCID: PMC4084881  PMID: 20381196
Mutation; rare variation; genetics; association

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