Characterizing the Major Structural Variant Alleles of the Human Genome.

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity.

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution.

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis.

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.

Long-read sequence and assembly of segmental duplications.

We have developed a computational method based on polyploid phasing of long sequence reads to resolve collapsed regions of segmental duplications within genome assemblies. Segmental Duplication Assembler (SDA; https://github.com/mvollger/SDA ) constructs graphs in which paralogous sequence variants define the nodes and long-read sequences provide attraction and repulsion edges, enabling the partition and assembly of long reads corresponding to distinct paralogs. We apply it to single-molecule, real-time sequence data from three human genomes and recover 33-79 megabase pairs (Mb) of duplications in which approximately half of the loci are diverged (<99.8%) compared to the reference genome. We show that the corresponding sequence is highly accurate (>99.9%) and that the diverged sequence corresponds to copy-number-variable paralogs that are absent from the human reference genome. Our method can be applied to other complex genomes to resolve the last gene-rich gaps, improve duplicate gene annotation, and better understand copy-number-variant genetic diversity at the base-pair level.

Transcriptional fates of human-specific segmental duplications in brain.

Despite the importance of duplicate genes for evolutionary adaptation, accurate gene annotation is often incomplete, incorrect, or lacking in regions of segmental duplication. We developed an approach combining long-read sequencing and hybridization capture to yield full-length transcript information and confidently distinguish between nearly identical genes/paralogs. We used biotinylated probes to enrich for full-length cDNA from duplicated regions, which were then amplified, size-fractionated, and sequenced using single-molecule, long-read sequencing technology, permitting us to distinguish between highly identical genes by virtue of multiple paralogous sequence variants. We examined 19 gene families as expressed in developing and adult human brain, selected for their high sequence identity (average >99%) and overlap with human-specific segmental duplications (SDs). We characterized the transcriptional differences between related paralogs to better understand the birth-death process of duplicate genes and particularly how the process leads to gene innovation. In 48% of the cases, we find that the expressed duplicates have changed substantially from their ancestral models due to novel sites of transcription initiation, splicing, and polyadenylation, as well as fusion transcripts that connect duplication-derived exons with neighboring genes. We detect unannotated open reading frames in genes currently annotated as pseudogenes, while relegating other duplicates to nonfunctional status. Our method significantly improves gene annotation, specifically defining full-length transcripts, isoforms, and open reading frames for new genes in highly identical SDs. The approach will be more broadly applicable to genes in structurally complex regions of other genomes where the duplication process creates novel genes important for adaptive traits.

Multi-scale visual analysis of time-varying electrocorticography data via clustering of brain regions.

BACKGROUND: There exists a need for effective and easy-to-use software tools supporting the analysis of complex Electrocorticography (ECoG) data. Understanding how epileptic seizures develop or identifying diagnostic indicators for neurological diseases require the in-depth analysis of neural activity data from ECoG. Such data is multi-scale and is of high spatio-temporal resolution. Comprehensive analysis of this data should be supported by interactive visual analysis methods that allow a scientist to understand functional patterns at varying levels of granularity and comprehend its time-varying behavior. RESULTS: We introduce a novel multi-scale visual analysis system, ECoG ClusterFlow, for the detailed exploration of ECoG data. Our system detects and visualizes dynamic high-level structures, such as communities, derived from the time-varying connectivity network. The system supports two major views: 1) an overview summarizing the evolution of clusters over time and 2) an electrode view using hierarchical glyph-based design to visualize the propagation of clusters in their spatial, anatomical context. We present case studies that were performed in collaboration with neuroscientists and neurosurgeons using simulated and recorded epileptic seizure data to demonstrate our system's effectiveness. CONCLUSION: ECoG ClusterFlow supports the comparison of spatio-temporal patterns for specific time intervals and allows a user to utilize various clustering algorithms. Neuroscientists can identify the site of seizure genesis and its spatial progression during various the stages of a seizure. Our system serves as a fast and powerful means for the generation of preliminary hypotheses that can be used as a basis for subsequent application of rigorous statistical methods, with the ultimate goal being the clinical treatment of epileptogenic zones.

Distinct requirements for wnt9a and irf6 in extension and integration mechanisms during zebrafish palate morphogenesis.

Development of the palate in vertebrates involves cranial neural crest migration, convergence of facial prominences and extension of the cartilaginous framework. Dysregulation of palatogenesis results in orofacial clefts, which represent the most common structural birth defects. Detailed analysis of zebrafish palatogenesis revealed distinct mechanisms of palatal morphogenesis: extension, proliferation and integration. We show that wnt9a is required for palatal extension, wherein the chondrocytes form a proliferative front, undergo morphological change and intercalate to form the ethmoid plate. Meanwhile, irf6 is required specifically for integration of facial prominences along a V-shaped seam. This work presents a mechanistic analysis of palate morphogenesis in a clinically relevant context.

Requirement for frzb and fzd7a in cranial neural crest convergence and extension mechanisms during zebrafish palate and jaw morphogenesis.

Regulation of convergence and extension by wnt-frizzled signaling is a common theme in embryogenesis. This study examines the functional requirements of frzb and fzd7a in convergence and extension mechanisms during craniofacial development. Using a morpholino knockdown approach, we found that frzb and fzd7a are dispensable for directed migration of the bilateral trabeculae, but necessary for the convergence and extension of the palatal elements, where the extension process is mediated by chondrocyte proliferation, morphologic change and intercalation. In contrast, frzb and fzd7a are required for convergence of the mandibular prominences, where knockdown of either frzb or fzd7a resulted in complete loss of lower jaw structures. Further, we found that bapx1 was specifically downregulated in the wnt9a/frzb/fzd7a morphants, while general neural crest markers were unaffected. In addition, expression of wnt9a and frzb was also absent in the edn-/- mutant. Notably, over-expression of bapx1 was sufficient to partially rescue mandibular elements in the wnt9a/frzb/fzd7a morphants, demonstrating genetic epistasis of bapx1 acting downstream of edn1 and wnt9a/frzb/fzd7a in lower jaw development. This study underscores the important role of wnt-frizzled signaling in convergence and extension in palate and craniofacial morphogenesis, distinct regulation of upper vs. lower jaw structures, and integration of wnt-frizzled with endothelin signaling to coordinate shaping of the facial form.

Independent expansion, selection and hypervariability of the TBC1D3 gene family in humans.

TBC1D3 is a primate-specific gene family that has expanded in the human lineage and has been implicated in neuronal progenitor proliferation and expansion of the frontal cortex. The gene family and its expression have been challenging to investigate because it is embedded in high-identity and highly variable segmental duplications. We sequenced and assembled the gene family using long-read sequencing data from 34 humans and 11 nonhuman primate species. Our analysis shows that this particular gene family has independently duplicated in at least five primate lineages, and the duplicated loci are enriched at sites of large-scale chromosomal rearrangements on chromosome 17. We find that most humans vary along two TBC1D3 clusters where human haplotypes are highly variable in copy number, differing by as many as 20 copies, and structure (structural heterozygosity 90%). We also show evidence of positive selection, as well as a significant change in the predicted human TBC1D3 protein sequence. Lastly, we find that, despite multiple duplications, human TBC1D3 expression is limited to a subset of copies and, most notably, from a single paralog group: TBC1D3-CDKL. These observations may help explain why a gene potentially important in cortical development can be so variable in the human population.

Clonal hematopoiesis and inflammation: A review of mechanisms and clinical implications.

Clonal hematopoiesis (CH) is defined by the presence of somatic mutations in hematopoietic stem and progenitor cells (HSPC). CH is associated primarily with advancing age and confers an elevated risk of progression to overt hematologic malignancy and cardiovascular disease. Increasingly, CH is associated with a wide range of diseases driven by, and sequelae of, inflammation. Accordingly, there is great interest in better understanding the pathophysiologic and clinical relationship between CH, aging, and disease. Both observational and experimental findings support the concept that CH is a potential common denominator in the inflammatory outcomes of aging. However, there is also evidence that local and systemic inflammatory states promote the growth and select for CH clones. In this review, we aim to provide an up-to-date summary of the nature of the relationship between inflammation and CH, which is central to unlocking potential therapeutic opportunities to prevent progression to myeloid malignancy.

Embryonic fate map of first pharyngeal arch structures in the sox10: kaede zebrafish transgenic model.

Cranial neural crest cells follow stereotypic patterns of migration to form craniofacial structures. The zebrafish is a powerful vertebrate genetic model where transgenics with reporter proteins under the transcriptional regulation of lineage-specific promoters can be generated. Numerous studies demonstrate that the zebrafish ethmoid plate is embryologically analogous to the mammalian palate. A fate map correlating embryonic cranial neural crest to defined jaw structures would provide a useful context for the morphogenetic analysis of craniofacial development. To that end, the sox10:kaede transgenic was generated, where sox10 provides lineage restriction to the neural crest. Specific regions of neural crest were labeled at the 10-somite stage by photoconversion of the kaede reporter protein. Lineage analysis was carried out during pharyngeal development in wild-type animals, after miR140 injection, and after estradiol treatment. At the 10-somite stage, cranial neural crest cells anterior of the eye contributed to the median ethmoid plate, whereas cells medial to the eye formed the lateral ethmoid plate and trabeculae and a posterior population formed the mandible. miR-140 overexpression and estradiol inhibition of Hedgehog signaling resulted in cleft development, with failed migration of the anterior cell population to form the median ethmoid plate. The sox10:kaede transgenic line provides a useful tool for neural crest lineage analysis. These studies illustrate the advantages of the zebrafish model for application in morphogenetic studies of vertebrate craniofacial development.

An evolutionary driver of interspersed segmental duplications in primates.

BACKGROUND: The complex interspersed pattern of segmental duplications in humans is responsible for rearrangements associated with neurodevelopmental disease, including the emergence of novel genes important in human brain evolution. We investigate the evolution of LCR16a, a putative driver of this phenomenon that encodes one of the most rapidly evolving human-ape gene families, nuclear pore interacting protein (NPIP). RESULTS: Comparative analysis shows that LCR16a has independently expanded in five primate lineages over the last 35 million years of primate evolution. The expansions are associated with independent lineage-specific segmental duplications flanking LCR16a leading to the emergence of large interspersed duplication blocks at non-orthologous chromosomal locations in each primate lineage. The intron-exon structure of the NPIP gene family has changed dramatically throughout primate evolution with different branches showing characteristic gene models yet maintaining an open reading frame. In the African ape lineage, we detect signatures of positive selection that occurred after a transition to more ubiquitous expression among great ape tissues when compared to Old World and New World monkeys. Mouse transgenic experiments from baboon and human genomic loci confirm these expression differences and suggest that the broader ape expression pattern arose due to mutational changes that emerged in cis. CONCLUSIONS: LCR16a promotes serial interspersed duplications and creates hotspots of genomic instability that appear to be an ancient property of primate genomes. Dramatic changes to NPIP gene structure and altered tissue expression preceded major bouts of positive selection in the African ape lineage, suggestive of a gene undergoing strong adaptive evolution.

High-resolution comparative analysis of great ape genomes.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.

The birth of a human-specific neural gene by incomplete duplication and gene fusion.

BACKGROUND: Gene innovation by duplication is a fundamental evolutionary process but is difficult to study in humans due to the large size, high sequence identity, and mosaic nature of segmental duplication blocks. The human-specific gene hydrocephalus-inducing 2, HYDIN2, was generated by a 364 kbp duplication of 79 internal exons of the large ciliary gene HYDIN from chromosome 16q22.2 to chromosome 1q21.1. Because the HYDIN2 locus lacks the ancestral promoter and seven terminal exons of the progenitor gene, we sought to characterize transcription at this locus by coupling reverse transcription polymerase chain reaction and long-read sequencing. RESULTS: 5' RACE indicates a transcription start site for HYDIN2 outside of the duplication and we observe fusion transcripts spanning both the 5' and 3' breakpoints. We observe extensive splicing diversity leading to the formation of altered open reading frames (ORFs) that appear to be under relaxed selection. We show that HYDIN2 adopted a new promoter that drives an altered pattern of expression, with highest levels in neural tissues. We estimate that the HYDIN duplication occurred ~3.2 million years ago and find that it is nearly fixed (99.9%) for diploid copy number in contemporary humans. Examination of 73 chromosome 1q21 rearrangement patients reveals that HYDIN2 is deleted or duplicated in most cases. CONCLUSIONS: Together, these data support a model of rapid gene innovation by fusion of incomplete segmental duplications, altered tissue expression, and potential subfunctionalization or neofunctionalization of HYDIN2 early in the evolution of the Homo lineage.

Methods for Specifying Scientific Data Standards and Modeling Relationships with Applications to Neuroscience.

Neuroscience continues to experience a tremendous growth in data; in terms of the volume and variety of data, the velocity at which data is acquired, and in turn the veracity of data. These challenges are a serious impediment to sharing of data, analyses, and tools within and across labs. Here, we introduce BRAINformat, a novel data standardization framework for the design and management of scientific data formats. The BRAINformat library defines application-independent design concepts and modules that together create a general framework for standardization of scientific data. We describe the formal specification of scientific data standards, which facilitates sharing and verification of data and formats. We introduce the concept of Managed Objects, enabling semantic components of data formats to be specified as self-contained units, supporting modular and reusable design of data format components and file storage. We also introduce the novel concept of Relationship Attributes for modeling and use of semantic relationships between data objects. Based on these concepts we demonstrate the application of our framework to design and implement a standard format for electrophysiology data and show how data standardization and relationship-modeling facilitate data analysis and sharing. The format uses HDF5, enabling portable, scalable, and self-describing data storage and integration with modern high-performance computing for data-driven discovery. The BRAINformat library is open source, easy-to-use, and provides detailed user and developer documentation and is freely available at: https://bitbucket.org/oruebel/brainformat.

Genome sequencing in a case of Niemann-Pick type C.

Adult-onset Niemann-Pick disease type C (NPC) is an infrequent presentation of a rare neurovisceral lysosomal lipid storage disorder caused by autosomal recessive mutations in NPC1 (∼95%) or NPC2 (∼5%). Our patient was diagnosed at age 33 when he presented with a 10-yr history of difficulties in judgment, concentration, speech, and coordination. A history of transient neonatal jaundice and splenomegaly with bone marrow biopsy suggesting a lipid storage disorder pointed to NPC; biochemical ("variant" level cholesterol esterification) and ultrastructural studies in adulthood confirmed the diagnosis. Genetic testing revealed two different missense mutations in the NPC1 gene-V950M and N1156S. Symptoms progressed over >20 yr to severe ataxia and spasticity, dementia, and dysphagia with aspiration leading to death. Brain autopsy revealed mild atrophy of the cerebrum and cerebellum. Microscopic examination showed diffuse gray matter deposition of balloon neurons, mild white matter loss, extensive cerebellar Purkinje cell loss with numerous "empty baskets," and neurofibrillary tangles predominantly in the hippocampal formation and transentorhinal cortex. We performed whole-genome sequencing to examine whether the patient harbored variants outside of the NPC1 locus that could have contributed to his late-onset phenotype. We focused analysis on genetic modifiers in pathways related to lipid metabolism, longevity, and neurodegenerative disease. We identified no rare coding variants in any of the pathways examined nor was the patient enriched for genome-wide association study (GWAS) single-nucleotide polymorphisms (SNPs) associated with longevity or altered lipid metabolism. In light of these findings, this case provides support for the V950M variant being sufficient for adult-onset NPC disease.

Neural crest development and craniofacial morphogenesis is coordinated by nitric oxide and histone acetylation.

Cranial neural crest (CNC) cells are patterned and coalesce to facial prominences that undergo convergence and extension to generate the craniofacial form. We applied a chemical genetics approach to identify pathways that regulate craniofacial development during embryogenesis. Treatment with the nitric oxide synthase inhibitor 1-(2-[trifluoromethyl] phenyl) imidazole (TRIM) abrogated first pharyngeal arch structures and induced ectopic ceratobranchial formation. TRIM promoted a progenitor CNC fate and inhibited chondrogenic differentiation, which were mediated through impaired nitric oxide (NO) production without appreciable effect on global protein S-nitrosylation. Instead, TRIM perturbed hox gene patterning and caused histone hypoacetylation. Rescue of TRIM phenotype was achieved with overexpression of histone acetyltransferase kat6a, inhibition of histone deacetylase, and complementary NO. These studies demonstrate that NO signaling and histone acetylation are coordinated mechanisms that regulate CNC patterning, differentiation, and convergence during craniofacial morphogenesis.

Genome sequencing of idiopathic pulmonary fibrosis in conjunction with a medical school human anatomy course.

Even in cases where there is no obvious family history of disease, genome sequencing may contribute to clinical diagnosis and management. Clinical application of the genome has not yet become routine, however, in part because physicians are still learning how best to utilize such information. As an educational research exercise performed in conjunction with our medical school human anatomy course, we explored the potential utility of determining the whole genome sequence of a patient who had died following a clinical diagnosis of idiopathic pulmonary fibrosis (IPF). Medical students performed dissection and whole genome sequencing of the cadaver. Gross and microscopic findings were more consistent with the fibrosing variant of nonspecific interstitial pneumonia (NSIP), as opposed to IPF per se. Variants in genes causing Mendelian disorders predisposing to IPF were not detected. However, whole genome sequencing identified several common variants associated with IPF, including a single nucleotide polymorphism (SNP), rs35705950, located in the promoter region of the gene encoding mucin glycoprotein MUC5B. The MUC5B promoter polymorphism was recently found to markedly elevate risk for IPF, though a particular association with NSIP has not been previously reported, nor has its contribution to disease risk previously been evaluated in the genome-wide context of all genetic variants. We did not identify additional predicted functional variants in a region of linkage disequilibrium (LD) adjacent to MUC5B, nor did we discover other likely risk-contributing variants elsewhere in the genome. Whole genome sequencing thus corroborates the association of rs35705950 with MUC5B dysregulation and interstitial lung disease. This novel exercise additionally served a unique mission in bridging clinical and basic science education.

Visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy.

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.