Ethical Considerations of Genome Sequencing for Pediatric Patients.

Advancements in genetic testing in the healthcare setting, most recently genomic sequencing, has enhanced our ability to diagnose genetic conditions. These advances include increased accessibility and affordability of genomic technologies. With expanded use comes the potential for significant ethical challenges for clinicians, particularly considering the implications of testing a child for one condition and incidentally finding a different condition or health risk. In this focused review, we address various ethical considerations from informed consent to the rights of a child undergoing genetic testing.

Survival in elderly patients with breast cancer with and without autoimmune disease.

BACKGROUND: Patients with certain autoimmune conditions are at a reduced risk of developing breast cancer compared to the general population. Despite this, little is known about outcomes in patients with breast cancer who have a concurrent autoimmune diagnosis. METHODS: This study compared differences in outcomes between women with breast cancer who had or did not have an autoimmune diagnosis. The SEER-Medicare databases (2007-2014) were used to identify patients with breast cancer and diagnosis codes were used to identify those with an autoimmune disorder. RESULTS: The studied autoimmune diseases had a prevalence of 27% among the 137,324 patients with breast cancer. Autoimmune disease was associated with significantly longer overall survival (OS) and significantly lower cancer-specific mortality (CSM) among stage IV breast cancer patients (p < 0.0001). After controlling for the effects of age, race, chronic kideny disease, chemotherapy, and radiation therapy autoimmune disease was still predictive of improved OS (HR: 1.45, 95% CI: 1.35-1.55, p < 0.0001) and CSM (HR: 1.40, 95% CI: 1.29-1.5, p < 0.0001). By contrast, in patients with stage I-III breast cancer, the presence of an autoimmune diagnosis was associated with a lower OS (p < 0.0001, p < 0.0001, and p = 0.026, respectively), compared to patients without autoimmune disease. CONCLUSIONS: We found a higher prevalence of rheumatoid arthritis, Crohn's disease, ulcerative colitis, and systemic lupus erythematosus in patients with breast cancer compared to age matched cohorts in the general population. The presence of an autoimmune diagnosis was associated with a lower OS in stages I-III breast cancer and improved OS and CSM in patients with stage IV disease. These results suggest that anti-tumor immunity plays an important role in late stage breast cancer and could potentially be exploited to improve the effectiveness of immunotherapy.

Comparative analysis of genome-scale, base-resolution DNA methylation profiles across 580 animal species.

Methylation of cytosines is a prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms across the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale DNA methylation profiles of multiple organs. Bioinformatic analysis of this large dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions-once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution.

Comparing Survival in Patients With Lung Cancer With and Without a History of Common Autoimmune Disease.

Introduction: Autoimmune disease has both a predisposing and a protective effect toward malignancy. Though studies have investigated the risk of malignancy in patients with autoimmune disease, there is limited research on how autoimmunity affects survival. Methods: This study compared survival in patients with lung cancer with and without autoimmune disease. Patients with lung cancer were culled from the Surveillance, Epidemiology, and End Results Medicare databases (2007-2014), and autoimmune diseases were identified using diagnosis codes. Results: The overall prevalence of investigated autoimmune diseases among the 112,445 patients was 22.7%. Overall survival (OS) (p < 0.0001) was longer and cancer-specific mortality (CSM) (p < 0.0001) reduced among patients with autoimmune disease. Median OS was 5 months higher. Improved OS and CSM were also apparent in disease stages 1, 3, and 4 in the NSCLC and SCLC subgroups (p < 0.0001) and across most specific autoimmune diseases. After adjusting for the effects of age, sex, race, disease stage, and chronic kidney disease, autoimmune disease was still predictive of higher OS (hazard ratio = 1.23, 95% confidence interval: 1.21-1.25, p < 0.0001) and reduced CSM (hazard ratio = 1.16, 95% confidence interval: 1.14-1.18, p < 0.0001). Conclusions: The prevalence of rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematous was highly enriched compared with the general population. The improvement in OS and CSM was larger in NSCLC than in SCLC, suggesting a larger role for the immune system in NSCLC. Alternate explanations for the improved survival include lead time bias, better access to health care, and a survival or autoimmunity-inducing genetic factor.

The giant axolotl genome uncovers the evolution, scaling, and transcriptional control of complex gene loci.

Vertebrates harbor recognizably orthologous gene complements but vary 100-fold in genome size. How chromosomal organization scales with genome expansion is unclear, and how acute changes in gene regulation, as during axolotl limb regeneration, occur in the context of a vast genome has remained a riddle. Here, we describe the chromosome-scale assembly of the giant, 32 Gb axolotl genome. Hi-C contact data revealed the scaling properties of interphase and mitotic chromosome organization. Analysis of the assembly yielded understanding of the evolution of large, syntenic multigene clusters, including the Major Histocompatibility Complex (MHC) and the functional regulatory landscape of the Fibroblast Growth Factor 8 (Axfgf8) region. The axolotl serves as a primary model for studying successful regeneration.

Characterization of axolotl lampbrush chromosomes by fluorescence in situ hybridization and immunostaining.

The lampbrush chromosomes (LBCs) in oocytes of the Mexican axolotl (Ambystoma mexicanum) were identified some time ago by their relative lengths and predicted centromeres, but they have never been associated completely with the mitotic karyotype, linkage maps or genome assembly. We identified 9 of the axolotl LBCs using RNAseq to identify actively transcribed genes and 13 BAC (bacterial artificial clone) probes containing pieces of active genes. Using read coverage analysis to find candidate centromere sequences, we developed a centromere probe that localizes to all 14 centromeres. Measurements of relative LBC arm lengths and polymerase III localization patterns enabled us to identify all LBCs. This study presents a relatively simple and reliable way to identify each axolotl LBC cytologically and to anchor chromosome-length sequences (from the axolotl genome assembly) to the physical LBCs by immunostaining and fluorescence in situ hybridization. Our data will facilitate a more detailed transcription analysis of individual LBC loops.

Spinal Muscular Atrophy: Mutations, Testing, and Clinical Relevance.

Spinal muscular atrophy (SMA) is a heritable neuromuscular disorder that causes degeneration of the alpha motor neurons from anterior horn cells in the spinal cord, which causes severe progressive hypotonia and muscular weakness. With a carrier frequency of 1 in 40-50 and an estimated incidence of 1 in 10,000 live births, SMA is the second most common autosomal recessive disorder. Affected individuals with SMA have a homozygous loss of function of the survival motor neuron gene SMN1 on 5q13 but keep the modifying SMN2 gene. The most common mutation causing SMA is a homozygous deletion of the SMN1 exon 7, which can be readily detected and used as a sensitive diagnostic test. Because SMN2 produces a reduced number of full-length transcripts, the number of SMN2 copies can modify the clinical phenotype and as such, becomes an essential predictive factor. Population-based SMA carrier screening identifies carrier couples that may pass on this genetic disorder to their offspring and allows the carriers to make informed reproductive choices or prepare for immediate treatment for an affected child. Three treatments have recently been approved by the Food and Drug Administration (FDA). Nusinersen increases the expression levels of the SMN protein using an antisense oligonucleotide to alter splicing of the SMN2 transcript. Onasemnogene abeparvovec is a gene therapy that utilizes an adeno-associated virus serotype 9 vector to increase low functional SMN protein levels. Risdiplam is a small molecule that alters SMN2 splicing in order to increase functional SMN protein. Newborn screening for SMA has been shown to be successful in allowing infants to be treated before the loss of motor neurons and has resulted in improved clinical outcomes. Several of the recommendations and guidelines in the review are based on studies performed in the United States.

A chromosome-scale assembly of the axolotl genome.

The axolotl (Ambystoma mexicanum) provides critical models for studying regeneration, evolution, and development. However, its large genome (∼32 Gb) presents a formidable barrier to genetic analyses. Recent efforts have yielded genome assemblies consisting of thousands of unordered scaffolds that resolve gene structures, but do not yet permit large-scale analyses of genome structure and function. We adapted an established mapping approach to leverage dense SNP typing information and for the first time assemble the axolotl genome into 14 chromosomes. Moreover, we used fluorescence in situ hybridization to verify the structure of these 14 scaffolds and assign each to its corresponding physical chromosome. This new assembly covers 27.3 Gb and encompasses 94% of annotated gene models on chromosomal scaffolds. We show the assembly's utility by resolving genome-wide orthologies between the axolotl and other vertebrates, identifying the footprints of historical introgression events that occurred during the development of axolotl genetic stocks, and precisely mapping several phenotypes including a large deletion underlying the cardiac mutant. This chromosome-scale assembly will greatly facilitate studies of the axolotl in biological research.

Miniscule differences between sex chromosomes in the giant genome of a salamander.

In the Mexican axolotl (Ambystoma mexicanum), sex is determined by a single Mendelian factor, yet its sex chromosomes do not exhibit morphological differentiation typical of many vertebrate taxa that possess a single sex-determining locus. As sex chromosomes are theorized to differentiate rapidly, species with undifferentiated sex chromosomes provide the opportunity to reconstruct early events in sex chromosome evolution. Whole genome sequencing of 48 salamanders, targeted chromosome sequencing and in situ hybridization were used to identify the homomorphic sex chromosome that carries an A. mexicanum sex-determining factor and sequences that are present only on the W chromosome. Altogether, these sequences cover ~300 kb of validated female-specific (W chromosome) sequence, representing ~1/100,000th of the 32 Gb genome. Notably, a recent duplication of ATRX, a gene associated with mammalian sex-determining pathways, is one of few functional (non-repetitive) genes identified among these W-specific sequences. This duplicated gene (ATRW) was used to develop highly predictive markers for diagnosing sex and represents a strong candidate for a recently-acquired sex determining locus (or sexually antagonistic gene) in A. mexicanum.

Publisher Correction: The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution.

When published, this article did not initially appear open access. This error has been corrected, and the open access status of the paper is noted in all versions of the paper. Additionally, affiliation 16 denoting equal contribution was missing from author Robb Krumlauf in the PDF originally published. This error has also been corrected.

Publisher Correction: The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution.

In the version of this article initially published, the present addresses for authors Dorit Hockman and Chris Amemiya were switched. The error has been corrected in the HTML and PDF versions of the article.

The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution.

The sea lamprey (Petromyzon marinus) serves as a comparative model for reconstructing vertebrate evolution. To enable more informed analyses, we developed a new assembly of the lamprey germline genome that integrates several complementary data sets. Analysis of this highly contiguous (chromosome-scale) assembly shows that both chromosomal and whole-genome duplications have played significant roles in the evolution of ancestral vertebrate and lamprey genomes, including chromosomes that carry the six lamprey HOX clusters. The assembly also contains several hundred genes that are reproducibly eliminated from somatic cells during early development in lamprey. Comparative analyses show that gnathostome (mouse) homologs of these genes are frequently marked by polycomb repressive complexes (PRCs) in embryonic stem cells, suggesting overlaps in the regulatory logic of somatic DNA elimination and bivalent states that are regulated by early embryonic PRCs. This new assembly will enhance diverse studies that are informed by lampreys' unique biology and evolutionary/comparative perspective.

A linkage map for the Newt Notophthalmus viridescens: Insights in vertebrate genome and chromosome evolution.

Genetic linkage maps are fundamental resources that enable diverse genetic and genomic approaches, including quantitative trait locus (QTL) analyses and comparative studies of genome evolution. It is straightforward to build linkage maps for species that are amenable to laboratory culture and genetic crossing designs, and that have relatively small genomes and few chromosomes. It is more difficult to generate linkage maps for species that do not meet these criteria. Here, we introduce a method to rapidly build linkage maps for salamanders, which are known for their enormous genome sizes. As proof of principle, we developed a linkage map with thousands of molecular markers (N=2349) for the Eastern newt (Notophthalmus viridescens). The map contains 12 linkage groups (152.3-934.7cM), only one more than the number of chromosome pairs. Importantly, this map was generated using RNA isolated from a single wild caught female and her 28 offspring. We used the map to reveal chromosome-scale conservation of synteny among N. viridescens, A. mexicanum (Urodela), and chicken (Amniota), and to identify large conserved segments between N. viridescens and Xenopus tropicalis (Anura). We also show that met1, a major effect QTL that regulates the expression of alternate metamorphic and paedomorphic modes of development in Ambystoma, associates with a chromosomal fusion that is not found in the N. viridescens map. Our results shed new light on the ancestral amphibian karyotype and reveal specific fusion and translocation events that shaped the genomes of three amphibian model taxa. The ability to rapidly build linkage maps for large salamander genomes will enable genetic and genomic analyses within this important vertebrate group, and more generally, empower comparative studies of vertebrate biology and evolution.

Cellular and Molecular Features of Developmentally Programmed Genome Rearrangement in a Vertebrate (Sea Lamprey: Petromyzon marinus).

The sea lamprey (Petromyzon marinus) represents one of the few vertebrate species known to undergo large-scale programmatic elimination of genomic DNA over the course of its normal development. Programmed genome rearrangements (PGRs) result in the reproducible loss of ~20% of the genome from somatic cell lineages during early embryogenesis. Studies of PGR hold the potential to provide novel insights related to the maintenance of genome stability during the cell cycle and coordination between mechanisms responsible for the accurate distribution of chromosomes into daughter cells, yet little is known regarding the mechanistic basis or cellular context of PGR in this or any other vertebrate lineage. Here we identify epigenetic silencing events that are associated with the programmed elimination of DNA and describe the spatiotemporal dynamics of PGR during lamprey embryogenesis. In situ analyses reveal that the earliest DNA methylation (and to some extent H3K9 trimethylation) events are limited to specific extranuclear structures (micronuclei) containing eliminated DNA. During early embryogenesis a majority of micronuclei (~60%) show strong enrichment for repressive chromatin modifications (H3K9me3 and 5meC). These analyses also led to the discovery that eliminated DNA is packaged into chromatin that does not migrate with somatically retained chromosomes during anaphase, a condition that is superficially similar to lagging chromosomes observed in some cancer subtypes. Closer examination of "lagging" chromatin revealed distributions of repetitive elements, cytoskeletal contacts and chromatin contacts that provide new insights into the cellular mechanisms underlying the programmed loss of these segments. Our analyses provide additional perspective on the cellular and molecular context of PGR, identify new structures associated with elimination of DNA and reveal that PGR is completed over the course of several successive cell divisions.

Initial characterization of the large genome of the salamander Ambystoma mexicanum using shotgun and laser capture chromosome sequencing.

Vertebrates exhibit substantial diversity in genome size, and some of the largest genomes exist in species that uniquely inform diverse areas of basic and biomedical research. For example, the salamander Ambystoma mexicanum (the Mexican axolotl) is a model organism for studies of regeneration, development and genome evolution, yet its genome is ~10× larger than the human genome. As part of a hierarchical approach toward improving genome resources for the species, we generated 600 Gb of shotgun sequence data and developed methods for sequencing individual laser-captured chromosomes. Based on these data, we estimate that the A. mexicanum genome is ~32 Gb. Notably, as much as 19 Gb of the A. mexicanum genome can potentially be considered single copy, which presumably reflects the evolutionary diversification of mobile elements that accumulated during an ancient episode of genome expansion. Chromosome-targeted sequencing permitted the development of assemblies within the constraints of modern computational platforms, allowed us to place 2062 genes on the two smallest A. mexicanum chromosomes and resolves key events in the history of vertebrate genome evolution. Our analyses show that the capture and sequencing of individual chromosomes is likely to provide valuable information for the systematic sequencing, assembly and scaffolding of large genomes.

The sea lamprey meiotic map improves resolution of ancient vertebrate genome duplications.

It is generally accepted that many genes present in vertebrate genomes owe their origin to two whole-genome duplications that occurred deep in the ancestry of the vertebrate lineage. However, details regarding the timing and outcome of these duplications are not well resolved. We present high-density meiotic and comparative genomic maps for the sea lamprey (Petromyzon marinus), a representative of an ancient lineage that diverged from all other vertebrates ∼550 million years ago. Linkage analyses yielded a total of 95 linkage groups, similar to the estimated number of germline chromosomes (1n ∼ 99), spanning a total of 5570.25 cM. Comparative mapping data yield strong support for the hypothesis that a single whole-genome duplication occurred in the basal vertebrate lineage, but do not strongly support a hypothetical second event. Rather, these comparative maps reveal several evolutionarily independent segmental duplications occurring over the last 600+ million years of chordate evolution. This refined history of vertebrate genome duplication should permit more precise investigations of vertebrate evolution.