Diane E. Dickel

Microduplications of 16p11.2 are associated with schizophrenia.

Recurrent microdeletions and microduplications of a 600-kb genomic region of chromosome 16p11.2 have been implicated in childhood-onset developmental disorders. We report the association of 16p11.2 microduplications with schizophrenia in two large cohorts. The microduplication was detected in 12/1,906 (0.63%) cases and 1/3,971 (0.03%) controls (P = 1.2 × 10−5, OR = 25.8) from the initial cohort, and in 9/2,645 (0.34%) cases and 1/2,420 (0.04%) controls (P = 0.022, OR = 8.3) of the replication cohort. The 16p11.2 microduplication was associated with a 14.5-fold increased risk of schizophrenia (95% CI (3.3, 62)) in the combined sample. A meta-analysis of datasets for multiple psychiatric disorders showed a significant association of the microduplication with schizophrenia (P = 4.8 × 10−7), bipolar disorder (P = 0.017) and autism (P = 1.9 × 10−7). In contrast, the reciprocal microdeletion was associated only with autism and developmental disorders (P = 2.3 × 10−13). Head circumference was larger in patients with the microdeletion than in patients with the microduplication (P = 0.0007).

A recurrent 16p12.1 microdeletion supports a two-hit model for severe developmental delay

We report the identification of a recurrent, 520-kb 16p12.1 microdeletion associated with childhood developmental delay. The microdeletion was detected in 20 of 11,873 cases compared with 2 of 8,540 controls (P = 0.0009, OR = 7.2) and replicated in a second series of 22 of 9,254 cases compared with 6 of 6,299 controls (P = 0.028, OR = 2.5). Most deletions were inherited, with carrier parents likely to manifest neuropsychiatric phenotypes compared to non-carrier parents (P = 0.037, OR = 6). Probands were more likely to carry an additional large copy-number variant when compared to matched controls (10 of 42 cases, P = 5.7 × 10−5, OR = 6.6). The clinical features of individuals with two mutations were distinct from and/or more severe than those of individuals carrying only the co-occurring mutation. Our data support a two-hit model in which the 16p12.1 microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large deletions or duplications. Analysis of other microdeletions with variable expressivity indicates that this two-hit model might be more generally applicable to neuropsychiatric disease.

Association studies of serotonin system candidate genes in early-onset obsessive-compulsive disorder.

BACKGROUND Family-based evidence for association at serotonin system genes SLC6A4, HTR1B, HTR2A, and brain-derived neurotrophic factor (BDNF) has been previously reported in obsessive-compulsive disorder (OCD). Early-onset OCD is a more familial form of the disorder. METHODS We used the transmission-disequilibrium test of association at common polymorphisms in each of these genes in 54 parent-child trios ascertained through probands with early-onset OCD. RESULTS No evidence for association was detected at any of the polymorphisms in the entire set of subjects. Nominally significant association was found at the HTR2A rs6311 polymorphism in subjects with tic disorder and OCD (p = .05), replicating a previous finding in Tourette syndrome and OCD. Nominally significant association was also found for the SLC6A4 HT transporter gene-linked polymorphic region (5-HTTLPR) polymorphism for female subjects (p = .03). Neither association would remain significant after statistical correction for multiple testing. Despite no individual study reporting replication, a pooled analysis of five replication studies of the SLC6A4 5-HTTLPR polymorphism supports association (p = .02). CONCLUSIONS Low power across individual association studies in OCD may lead to a false acceptance of the null hypothesis. Accumulation of evidence from multiple studies will be necessary to evaluate the potential role for these genes in contributing to susceptibility to OCD.

Reduced transcript expression of genes affected by inherited and de novo CNVs in autism.

Individuals with autism are more likely to carry rare inherited and de novo copy number variants (CNVs). However, further research is needed to establish which CNVs are causal and the mechanisms by which these CNVs influence autism. We examined genomic DNA of children with autism (N=41) and healthy controls (N=367) for rare CNVs using a high-resolution array comparative genomic hybridization platform. We show that individuals with autism are more likely to harbor rare CNVs as small as ∼10 kb, a threshold not previously detectable, and that CNVs in cases disproportionately affect genes involved in transcription, nervous system development, and receptor activity. We also show that a subset of genes that have known or suspected allele-specific or imprinting effects and are within rare-case CNVs may undergo loss of transcript expression. In particular, expression of CNTNAP2 and ZNF214 are decreased in probands compared with their unaffected transmitting parents. Furthermore, expression of PRODH and ARID1B, two genes affected by de novo CNVs, are decreased in probands compared with controls. These results suggest that for some genes affected by CNVs in autism, reduced transcript expression may be a mechanism of pathogenesis during neurodevelopment.

Five novel loci for inherited hearing loss mapped by SNP-based homozygosity profiles in Palestinian families

In communities with high rates of consanguinity and consequently high prevalence of recessive phenotypes, homozygosity mapping with SNP arrays is an effective approach for gene discovery. In 20 Palestinian kindreds with prelingual nonsyndromic hearing loss, we generated homozygosity profiles reflecting linkage to the phenotype. Family sizes ranged from small nuclear families with two affected children, one unaffected sibling, and parents to multigenerational kindreds with 12 affected relatives. By including unaffected parents and siblings and screening 250 K SNP arrays, even small nuclear families yielded informative profiles. In 14 families, we identified the allele responsible for hearing loss by screening a single candidate gene in the longest homozygous region. Novel alleles included missense, nonsense, and splice site mutations of CDH23, MYO7A, MYO15A, OTOF, PJVK, Pendrin/SLC26A4, TECTA, TMHS, and TMPRSS3, and a large genomic deletion of Otoancorin (OTOA). All point mutations were rare in the Palestinian population (zero carriers in 288 unrelated controls); the carrier frequency of the OTOA genomic deletion was 1%. In six families, we identified five genomic regions likely to harbor novel genes for human hearing loss on chromosomes 1p13.3 (DFNB82), 9p23–p21.2/p13.3–q21.13 (DFNB83), 12q14.3–q21.2 (DFNB84; two families), 14q23.1–q31.1, and 17p12–q11.2 (DFNB85).

Function-based identification of mammalian enhancers using site-specific integration

The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro–differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.

Germline Chd8 haploinsufficiency alters brain development in mouse.

The chromatin remodeling gene CHD8 represents a central node in neurodevelopmental gene networks implicated in autism. We examined the impact of germline heterozygous frameshift Chd8 mutation on neurodevelopment in mice. Chd8+/del5 mice displayed normal social interactions with no repetitive behaviors but exhibited cognitive impairment correlated with increased regional brain volume, validating that phenotypes of Chd8+/del5 mice overlap pathology reported in humans with CHD8 mutations. We applied network analysis to characterize neurodevelopmental gene expression, revealing widespread transcriptional changes in Chd8+/del5 mice across pathways disrupted in neurodevelopmental disorders, including neurogenesis, synaptic processes and neuroimmune signaling. We identified a co-expression module with peak expression in early brain development featuring dysregulation of RNA processing, chromatin remodeling and cell-cycle genes enriched for promoter binding by Chd8, and we validated increased neuronal proliferation and developmental splicing perturbation in Chd8+/del5 mice. This integrative analysis offers an initial picture of the consequences of Chd8 haploinsufficiency for brain development.

Functional anatomy of distant-acting mammalian enhancers

Transcriptional enhancers are a major class of functional element embedded in the vast non-coding portion of the human genome. Acting over large genomic distances, enhancers play critical roles in the tissue and cell type-specific regulation of genes, and there is mounting evidence that they contribute to the aetiology of many human diseases. Methods for genome-wide mapping of enhancer regions are now available, but the functional architecture contained within human enhancer elements remains unclear. Here, we review recent approaches aimed at understanding the functional anatomy of individual enhancer elements, using systematic qualitative and quantitative assessments of mammalian enhancer variants in cultured cells and in vivo. These studies provide direct insight into common architectural characteristics of enhancers including the presence of multiple transcription factor-binding sites and the mixture of both transcriptionally activating and repressing domains within the same enhancer. Despite such progress in understanding the functional composition of enhancers, the inherent complexities of enhancer anatomy continue to limit our ability to predict the impact of sequence changes on in vivo enhancer function. While providing an initial glimpse into the mutability of mammalian enhancers, these observations highlight the continued need for experimental enhancer assessment as genome sequencing becomes routine in the clinic.

Enhancer redundancy provides phenotypic robustness in mammalian development

Distant-acting tissue-specific enhancers, which regulate gene expression, vastly outnumber protein-coding genes in mammalian genomes, but the functional importance of this regulatory complexity remains unclear. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Unexpectedly, none of the ten deletions of individual enhancers caused noticeable changes in limb morphology. By contrast, the removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain and heart) uncovered more than one thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes that provides an effective regulatory buffer to prevent deleterious phenotypic consequences upon the loss of individual enhancers.

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

Significance In mammals, when and where a gene is transcribed are primarily regulated by the activity of regulatory DNA elements, or enhancers. Genetic mutation disrupting enhancer function is emerging as one of the major causes of human diseases. However, our knowledge remains limited about the location and activity of enhancers in the numerous and distinct cell types and tissues. Here, we develop a computational approach, regulatory element prediction based on tissue-specific local epigenetic marks (REPTILE), to precisely locate enhancers based on genome-wide DNA methylation and histone modification profiling. We systematically tested REPTILE on a variety of human and mouse cell types and tissues. Compared with existing methods, we found that enhancer predictions from REPTILE are more likely to be active in vivo and the predicted locations are more accurate. Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulatory element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared with existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types. REPTILE is available at https://github.com/yupenghe/REPTILE/.