Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.

Micrognathia, glossoptosis, and cleft palate comprise one of the most common malformation sequences, Robin sequence. It is a component of the TARP syndrome, talipes equinovarus, atrial septal defect, Robin sequence, and persistent left superior vena cava. This disorder is X-linked and severe, with apparently 100% pre- or postnatal lethality in affected males. Here we characterize a second family with TARP syndrome, confirm linkage to Xp11.23-q13.3, perform massively parallel sequencing of X chromosome exons, filter the results via a number of criteria including the linkage region, use a unique algorithm to characterize sequence changes, and show that TARP syndrome is caused by mutations in the RBM10 gene, which encodes RNA binding motif 10. We further show that this previously uncharacterized gene is expressed in midgestation mouse embryos in the branchial arches and limbs, consistent with the human phenotype. We conclude that massively parallel sequencing is useful to characterize large candidate linkage intervals and that it can be used successfully to allow identification of disease-causing gene mutations.

Comparisons of chromosome X and the autosomes can illuminate differences in the histories of males and females as well as shed light on the forces of natural selection. We compared the patterns of variation in these parts of the genome using two datasets that we assembled for this study that are both genomic in scale. Three independent analyses show that around the time of the dispersal of modern humans out of Africa, chromosome X experienced much more genetic drift than is expected from the pattern on the autosomes. This is not predicted by known episodes of demographic history, and we found no similar patterns associated with the dispersals into East Asia and Europe. We conclude that a sex-biased process that reduced the female effective population size, or an episode of natural selection unusually affecting chromosome X, was associated with the founding of non-African populations.

The Hawaiian honeycreepers are an avian adaptive radiation containing many endangered and extinct species. They display a dramatic range of phenotypic variation and are a model system for studies of evolution, conservation, disease dynamics and population genetics. Development of a genome-scale resources for this group would augment the quality of research focusing on Hawaiian honeycreepers and facilitate comparative avian genomic research.

No abstract available

Antibodies of the VRC01 class neutralize HIV-1, arise in diverse HIV-1-infected donors, and are potential templates for an effective HIV-1 vaccine. However, the stochastic processes that generate repertoires in each individual of >10(12) antibodies make elicitation of specific antibodies uncertain. Here we determine the ontogeny of the VRC01 class by crystallography and next-generation sequencing. Despite antibody-sequence differences exceeding 50%, antibody-gp120 cocrystal structures reveal VRC01-class recognition to be remarkably similar. B cell transcripts indicate that VRC01-class antibodies require few specific genetic elements, suggesting that naive-B cells with VRC01-class features are generated regularly by recombination. Virtually all of these fail to mature, however, with only a few-likely one-ancestor B cell expanding to form a VRC01-class lineage in each donor. Developmental similarities in multiple donors thus reveal the generation of VRC01-class antibodies to be reproducible in principle, thereby providing a framework for attempts to elicit similar antibodies in the general population.

Whole-exome sequencing (WES) is rapidly evolving into a tool of choice for rapid, and inexpensive identification of molecular genetic lesions within targeted regions of the human genome. While biases in WES coverage of nucleotides in targeted regions are recognized, it is not well understood how repetition of WES improves the interpretation of sequencing results in a clinical diagnostic setting.

Despite the tremendous drop in the cost of nucleotide sequencing in recent years, many research projects still utilize sequencing of pools containing multiple samples for the detection of sequence variants as a cost saving measure. Various software tools exist to analyze these pooled sequence data, yet little has been reported on the relative accuracy and ease of use of these different programs.

Gorillas are humans' closest living relatives after chimpanzees, and are of comparable importance for the study of human origins and evolution. Here we present the assembly and analysis of a genome sequence for the western lowland gorilla, and compare the whole genomes of all extant great ape genera. We propose a synthesis of genetic and fossil evidence consistent with placing the human-chimpanzee and human-chimpanzee-gorilla speciation events at approximately 6 and 10 million years ago. In 30% of the genome, gorilla is closer to human or chimpanzee than the latter are to each other; this is rarer around coding genes, indicating pervasive selection throughout great ape evolution, and has functional consequences in gene expression. A comparison of protein coding genes reveals approximately 500 genes showing accelerated evolution on each of the gorilla, human and chimpanzee lineages, and evidence for parallel acceleration, particularly of genes involved in hearing. We also compare the western and eastern gorilla species, estimating an average sequence divergence time 1.75 million years ago, but with evidence for more recent genetic exchange and a population bottleneck in the eastern species. The use of the genome sequence in these and future analyses will promote a deeper understanding of great ape biology and evolution.

The utility of induced pluripotent stem cells (iPSCs) as models to study diseases and as sources for cell therapy depends on the integrity of their genomes. Despite recent publications of DNA sequence variations in the iPSCs, the true scope of such changes for the entire genome is not clear. Here we report the whole-genome sequencing of three human iPSC lines derived from two cell types of an adult donor by episomal vectors. The vector sequence was undetectable in the deeply sequenced iPSC lines. We identified 1,058-1,808 heterozygous single-nucleotide variants (SNVs), but no copy-number variants, in each iPSC line. Six to twelve of these SNVs were within coding regions in each iPSC line, but ~50% of them are synonymous changes and the remaining are not selectively enriched for known genes associated with cancers. Our data thus suggest that episome-mediated reprogramming is not inherently mutagenic during integration-free iPSC induction.

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

We describe the establishment and analysis of a genus-wide comparative framework composed of 12 bacterial artificial chromosome fingerprint and end-sequenced physical maps representing the 10 genome types of Oryza aligned to the O. sativa ssp. japonica reference genome sequence. Over 932 Mb of end sequence was analyzed for repeats, simple sequence repeats, miRNA and single nucleotide variations, providing the most extensive analysis of Oryza sequence to date.

DNA methylation is an essential epigenetic process in mammals, intimately involved in gene regulation. Here we address the extent to which genetics, sex, and pregnancy influence genomic DNA methylation by intercrossing 2 inbred mouse strains, C57BL/6N and C3H/HeN, and analyzing DNA methylation in parents and offspring using whole-genome bisulfite sequencing. Differential methylation across genotype is detected at thousands of loci and is preserved on parental alleles in offspring. In comparison of autosomal DNA methylation patterns across sex, hundreds of differentially methylated regions are detected. Comparison of animals with different histories of pregnancy within our study reveals a CpG methylation pattern that is restricted to female animals that had borne offspring. Collectively, our results demonstrate the stability of CpG methylation across generations, clarify the interplay of epigenetics with genetics and sex, and suggest that CpG methylation may serve as an epigenetic record of life events in somatic tissues at loci whose expression is linked to the relevant biology.

Hypertension occurs at a higher rate in African Americans than in European Americans. Based on the assumption that causal variants are more frequently found on DNA segments inherited from the ancestral population with higher disease risk, we employed admixture mapping to identify genetic loci with excess local African ancestry associated with blood pressure. Chromosomal regions 1q21.2-21.3, 4p15.1, 19q12 and 20p13 were significantly associated with diastolic blood pressure (β = 5.28, -7.94, -6.82 and 5.89, P-value = 6.39E-04, 2.07E-04, 6.56E-05 and 5.04E-04, respectively); 1q21.2-21.3 and 19q12 were also significantly associated with mean arterial pressure (β = 5.86 and -6.40, P-value = 5.32E-04 and 6.37E-04, respectively). We further selected SNPs that had large allele frequency differences within these regions and tested their association with blood pressure. SNP rs4815428 was significantly associated with diastolic blood pressure after Bonferroni correction (β = -2.42, P-value = 9.57E-04), and it partially explained the admixture mapping signal at 20p13. SNPs rs771205 (β = -1.99, P-value = 3.37E-03), rs3126067, rs2184953 and rs58001094 (the latter three exhibit strong linkage disequilibrium, β = -2.3, P-value = 1.4E-03) were identified to be significantly associated with mean arterial pressure, and together they fully explained the admixture signal at 1q21.2-21.3. Although no SNP at 4p15.1 showed large ancestral allele frequency differences in our dataset, we detected association at low-frequency African-specific variants that mapped predominantly to the gene PCDH7, which is most highly expressed in aorta. Our results suggest that these regions may harbor genetic variants that contribute to the different prevalence of hypertension.

Hydractinia is a colonial marine hydroid that exhibits remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, H. symbiolongicarpus and H. echinata, placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from non-self.

Diminished ovarian function occurs early and is a primary cause for age-related decline in female fertility; however, its underlying mechanism remains unclear. This study investigated the roles that genome and epigenome structure play in age-related changes in gene expression and ovarian function, using human ovarian granulosa cells as an experimental system. DNA methylomes were compared between two groups of women with distinct age-related differences in ovarian functions, using both Methylated DNA Capture followed by Next Generation Sequencing (MethylCap-seq) and Reduced Representation Bisulfite Sequencing (RRBS); their transcriptomes were investigated using mRNA-seq. Significant, non-random changes in transcriptome and DNA methylome features are observed in human ovarian granulosa cells as women age and their ovarian functions deteriorate. The strongest correlations between methylation and the age-related changes in gene expression are not confined to the promoter region; rather, high densities of hypomethylated CpG-rich regions spanning the gene body are preferentially associated with gene down-regulation. This association is further enhanced where CpG regions are localized near the 3'-end of the gene. Such features characterize several genes crucial in age-related decline in ovarian function, most notably the AMH (Anti-Müllerian Hormone) gene. The genome-wide correlation between the density of hypomethylated intragenic and 3'-end regions and gene expression suggests previously unexplored mechanisms linking epigenome structure to age-related physiology and pathology.

Although RNA-Seq data provide unprecedented isoform-level expression information, detection of alternative isoform regulation (AIR) remains difficult, particularly when working with an incomplete transcript annotation. We introduce JunctionSeq, a new method that builds on the statistical techniques used by the well-established DEXSeq package to detect differential usage of both exonic regions and splice junctions. In particular, JunctionSeq is capable of detecting differential usage of novel splice junctions without the need for an additional isoform assembly step, greatly improving performance when the available transcript annotation is flawed or incomplete. JunctionSeq also provides a powerful and streamlined visualization toolset that allows bioinformaticians to quickly and intuitively interpret their results. We tested our method on publicly available data from several experiments performed on the rat pineal gland and Toxoplasma gondii, successfully detecting known and previously validated AIR genes in 19 out of 19 gene-level hypothesis tests. Due to its ability to query novel splice sites, JunctionSeq is still able to detect these differences even when all alternative isoforms for these genes were not included in the transcript annotation. JunctionSeq thus provides a powerful method for detecting alternative isoform regulation even with low-quality annotations. An implementation of JunctionSeq is available as an R/Bioconductor package.

Single point mutations at both synonymous and non-synonymous positions within exons can have severe effects on gene function through disruption of splicing. Predicting these mutations in silico purely from the genomic sequence is difficult due to an incomplete understanding of the multiple factors that may be responsible. In addition, little is known about which computational prediction approaches, such as those involving exonic splicing enhancers and exonic splicing silencers, are most informative.

The cat (Felis silvestris catus) shows significant variation in pelage, morphological, and behavioral phenotypes amongst its over 40 domesticated breeds. The majority of the breed specific phenotypic presentations originated through artificial selection, especially on desired novel phenotypic characteristics that arose only a few hundred years ago. Variations in coat texture and color of hair often delineate breeds amongst domestic animals. Although the genetic basis of several feline coat colors and hair lengths are characterized, less is known about the genes influencing variation in coat growth and texture, especially rexoid - curly coated types. Cornish Rex is a cat breed defined by a fixed recessive curly coat trait. Genome-wide analyses for selection (di, Tajima's D and nucleotide diversity) were performed in the Cornish Rex breed and in 11 phenotypically diverse breeds and two random bred populations. Approximately 63K SNPs were used in the analysis that aimed to localize the locus controlling the rexoid hair texture. A region with a strong signature of recent selective sweep was identified in the Cornish Rex breed on chromosome A1, as well as a consensus block of homozygosity that spans approximately 3 Mb. Inspection of the region for candidate genes led to the identification of the lysophosphatidic acid receptor 6 (LPAR6). A 4 bp deletion in exon 5, c.250_253_delTTTG, which induces a premature stop codon in the receptor, was identified via Sanger sequencing. The mutation is fixed in Cornish Rex, absent in all straight haired cats analyzed, and is also segregating in the German Rex breed. LPAR6 encodes a G protein-coupled receptor essential for maintaining the structural integrity of the hair shaft; and has mutations resulting in a wooly hair phenotype in humans.