The frequency of pathogenic variation in the All of Us cohort reveals ancestry-driven disparities.

Disparities in data underlying clinical genomic interpretation is an acknowledged problem, but there is a paucity of data demonstrating it. The All of Us Research Program is collecting data including whole-genome sequences, health records, and surveys for at least a million participants with diverse ancestry and access to healthcare, representing one of the largest biomedical research repositories of its kind. Here, we examine pathogenic and likely pathogenic variants that were identified in the All of Us cohort. The European ancestry subgroup showed the highest overall rate of pathogenic variation, with 2.26% of participants having a pathogenic variant. Other ancestry groups had lower rates of pathogenic variation, including 1.62% for the African ancestry group and 1.32% in the Latino/Admixed American ancestry group. Pathogenic variants were most frequently observed in genes related to Breast/Ovarian Cancer or Hypercholesterolemia. Variant frequencies in many genes were consistent with the data from the public gnomAD database, with some notable exceptions resolved using gnomAD subsets. Differences in pathogenic variant frequency observed between ancestral groups generally indicate biases of ascertainment of knowledge about those variants, but some deviations may be indicative of differences in disease prevalence. This work will allow targeted precision medicine efforts at revealed disparities.

Neptune: an environment for the delivery of genomic medicine.

PURPOSE: Genomic medicine holds great promise for improving health care, but integrating searchable and actionable genetic data into electronic health records (EHRs) remains a challenge. Here we describe Neptune, a system for managing the interaction between a clinical laboratory and an EHR system during the clinical reporting process. METHODS: We developed Neptune and applied it to two clinical sequencing projects that required report customization, variant reanalysis, and EHR integration. RESULTS: Neptune has been applied for the generation and delivery of over 15,000 clinical genomic reports. This work spans two clinical tests based on targeted gene panels that contain 68 and 153 genes respectively. These projects demanded customizable clinical reports that contained a variety of genetic data types including single-nucleotide variants (SNVs), copy-number variants (CNVs), pharmacogenomics, and polygenic risk scores. Two variant reanalysis activities were also supported, highlighting this important workflow. CONCLUSION: Methods are needed for delivering structured genetic data to EHRs. This need extends beyond developing data formats to providing infrastructure that manages the reporting process itself. Neptune was successfully applied on two high-throughput clinical sequencing projects to build and deliver clinical reports to EHR systems. The software is open source and available at https://gitlab.com/bcm-hgsc/neptune .

Whole genome detection of sequence and structural polymorphism in six diverse horses.

The domesticated horse has played a unique role in human history, serving not just as a source of animal protein, but also as a catalyst for long-distance migration and military conquest. As a result, the horse developed unique physiological adaptations to meet the demands of both their climatic environment and their relationship with man. Completed in 2009, the first domesticated horse reference genome assembly (EquCab 2.0) produced most of the publicly available genetic variations annotations in this species. Yet, there are around 400 geographically and physiologically diverse breeds of horse. To enrich the current collection of genetic variants in the horse, we sequenced whole genomes from six horses of six different breeds: an American Miniature, a Percheron, an Arabian, a Mangalarga Marchador, a Native Mongolian Chakouyi, and a Tennessee Walking Horse, and mapped them to EquCab3.0 genome. Aside from extreme contrasts in body size, these breeds originate from diverse global locations and each possess unique adaptive physiology. A total of 1.3 billion reads were generated for the six horses with coverage between 15x to 24x per horse. After applying rigorous filtration, we identified and functionally annotated 17,514,723 Single Nucleotide Polymorphisms (SNPs), and 1,923,693 Insertions/Deletions (INDELs), as well as an average of 1,540 Copy Number Variations (CNVs) and 3,321 Structural Variations (SVs) per horse. Our results revealed putative functional variants including genes associated with size variation like LCORL gene (found in all horses), ZFAT in the Arabian, American Miniature and Percheron horses and ANKRD1 in the Native Mongolian Chakouyi horse. We detected a copy number variation in the Latherin gene that may be the result of evolutionary selection impacting thermoregulation by sweating, an important component of athleticism and heat tolerance. The newly discovered variants were formatted into user-friendly browser tracks and will provide a foundational database for future studies of the genetic underpinnings of diverse phenotypes within the horse.

Mutation responsible for congenital photosensitivity and hyperbilirubinemia in Southdown sheep.

OBJECTIVE To identify the genetic cause for congenital photosensitivity and hyperbilirubinemia (CPH) in Southdown sheep. ANIMALS 73 Southdown sheep from a CPH research flock and 48 sheep of various breeds from commercial flocks without CPH. PROCEDURES Whole-genome sequencing was performed for a phenotypically normal Southdown sheep heterozygous for CPH. Heterozygous variants within Slco1b3 coding exons were identified, and exons that contained candidate mutations were amplified by PCR assay methods for Sanger sequencing. Blood samples from the other 72 Southdown sheep of the CPH research flock were used to determine plasma direct and indirect bilirubin concentrations. Southdown sheep with a plasma total bilirubin concentration < 0.3 mg/dL were classified as controls, and those with a total bilirubin concentration ≥ 0.3 mg/dL and signs of photosensitivity were classified as mutants. Sanger sequencing was used to determine the Slco1b3 genotype for all sheep. Genotypes were compared between mutants and controls of the CPH research flock and among all sheep. Protein homology was measured across 8 species to detect evolutionary conservation of Slco1b. RESULTS A nonsynonymous mutation at ovine Chr3:193,691,195, which generated a glycine-to-arginine amino acid change within the predicted Slco1b3 protein, was significantly associated with hyperbilirubinemia and predicted to be deleterious. That amino acid was conserved across 7 other mammalian species. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested a nonsynonymous mutation in Slco1b3 causes CPH in Southdown sheep. This disease appears to be similar to Rotor syndrome in humans. Sheep with CPH might be useful animals for Rotor syndrome research.

Complex disease and phenotype mapping in the domestic dog.

The domestic dog is becoming an increasingly valuable model species in medical genetics, showing particular promise to advance our understanding of cancer and orthopaedic disease. Here we undertake the largest canine genome-wide association study to date, with a panel of over 4,200 dogs genotyped at 180,000 markers, to accelerate mapping efforts. For complex diseases, we identify loci significantly associated with hip dysplasia, elbow dysplasia, idiopathic epilepsy, lymphoma, mast cell tumour and granulomatous colitis; for morphological traits, we report three novel quantitative trait loci that influence body size and one that influences fur length and shedding. Using simulation studies, we show that modestly larger sample sizes and denser marker sets will be sufficient to identify most moderate- to large-effect complex disease loci. This proposed design will enable efficient mapping of canine complex diseases, most of which have human homologues, using far fewer samples than required in human studies.

Kin discrimination and possible cryptic species in the social amoeba Polysphondylium violaceum.

BACKGROUND: The genetic diversity of many protists is unknown. The differences that result from this diversity can be important in interactions among individuals. The social amoeba Polysphondylium violaceum, which is a member of the Dictyostelia, has a social stage where individual amoebae aggregate together to form a multicellular fruiting body with dead stalk cells and live spores. Individuals can either cooperate with amoebae from the same clone, or sort to form clonal fruiting bodies. In this study we look at genetic diversity in P. violaceum and at how this diversity impacts social behavior. RESULTS: The phylogeny of the ribosomal DNA sequence (17S to 5.8S region) shows that P. violaceum is made up of at least two groups. Mating compatibility is more common between clones from the same phylogenetic group, though matings between clones from different phylogenetic groups sometimes occurred. P. violaceum clones are more likely to form clonal fruiting bodies when they are mixed with clones from a different group than when they are mixed with a clone of the same group. CONCLUSION: Both the phylogenetic and mating analyses suggest the possibility of cryptic species in P. violaceum. The level of divergence found within P. violaceum is comparable to the divergence between sibling species in other dictyostelids. Both major groups A/B and C/D/E/F show kin discrimination, which elevates relatedness within fruiting bodies but not to the level of clonality. The diminished cooperation in mixes between groups suggests that the level of genetic variation between individuals influences the extent of their cooperation.

Social evolution: kin preference in a social microbe.

Kin recognition helps cooperation to evolve in many animals, but it is uncertain whether microorganisms can also use it to focus altruistic behaviour on relatives. Here we show that the social amoeba Dictyostelium purpureum prefers to form groups with its own kin in situations where some individuals die to assist others. By directing altruism towards kin, D. purpureum should generally avoid the costs of chimaerism experienced by the related D. discoideum.

NO-dependent osteoclast motility: reliance on cGMP-dependent protein kinase I and VASP.

The osteoclast degrades bone in cycles; between cycles, the cell is motile. Resorption occurs by acid transport into an extracellular compartment defined by an alphavbeta3 integrin ring. NO has been implicated in the regulation of bone turnover due to stretch or via estrogen signals, but a specific mechanism linking NO to osteoclastic activity has not been described. NO stimulates osteoclast motility, and at high concentrations NO causes detachment and terminates resorption. Here we demonstrate that NO regulates attachment through the cGMP-dependent protein kinase I (PKG I) via phosphorylation of the intermediate protein VASP. VASP colocalized with the alphavbeta3 ring in stationary cells, but alternating bands of VASP and alphavbeta3 occurred when motility was induced by NO donors or cGMP. Redistribution of VASP correlated with its phosphorylation. Dependency of NO-induced motility on PKG I and on VASP was shown by siRNA knockdown of each protein. VASP knockdown also altered distribution of alphavbeta3 at the attachment site. We conclude that PKG I and VASP are essential for reorganization of attachment and cytoplasmic proteins in motility induced by NO or by cGMP.

Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.

Autosomal recessive osteopetrosis (ARO) is a paradigm for genetic diseases that cause severe, often irreversible, defects before birth. In ARO, osteoclasts cannot remove mineralized cartilage, bone marrow is severely reduced, and bone cannot be remodeled for growth. More than 50% of the patients show defects in the osteoclastic vacuolar-proton-pump subunit, ATP6a3. We treated ATP6a3-deficient mice by in utero heterologous hematopoietic stem cell (HSC) transplant from outbred GFP transgenic mice. Dramatic phenotype rescue by GFP osteoclasts was obtained with engraftment, which was observed in most cases. Engraftment survived for variable periods. Recipients were not immunosuppressed, and graft-versus-host disease was not observed in all pups born after in utero treatment. Thus, differentiation of unmatched HSC transplanted in utero is sufficient to prevent fatal defects in ARO and may prevent complications of ARO unresponsive to conventional bone marrow transplantation. The presence of defective cells is not a barrier to the rescue of the phenotype by donor HSC.

Negative regulation of RANKL-induced osteoclastic differentiation in RAW264.7 Cells by estrogen and phytoestrogens.

We studied estrogen effects on osteoclastic differentiation using RAW264.7, a murine monocytic cell line. Differentiation, in response to RANKL and colony-stimulating factor 1, was evaluated while varying estrogen receptor (ER) stimulation by estradiol or nonsteroidal ER agonists was performed. The RAW264.7 cells were found to express ERalpha but not ERbeta. In contrast to RANKL, which decreased ERalpha expression and induced osteoclast differentiation, 10 nm estradiol, 3 microm genistein, or 3 microm daidzein all increased ERalpha expression, stimulated cell proliferation, and decreased multinucleation, with the effects of estrogen > or = daidzein > genistein. However, no estrogen agonist reduced RANKL stimulation of osteoclast differentiation markers or its down-regulation of ERalpha expression by more than approximately 50%. Genistein is also an Src kinase antagonist in vitro, but it did not decrease Src phosphorylation in RAW264.7 cells relative to other estrogen agonists. However, both phytoestrogens and estrogen inhibited RANKL-induced IkappaB degradation and NF-kappaB nuclear localization with the same relative potency as seen in proliferation and differentiation assays. This study demonstrates, for the first time, the direct effects of estrogen on osteoclast precursor differentiation and shows that, in addition to effecting osteoblasts, estrogen may protect bone by reducing osteoclast production. Genistein, which activates ERs selectively, inhibited osteoclastogenesis less effectively than the nonselective phytoestrogen daidzein, which effectively reproduced effects of estrogen.

In vitro differentiation of CD14 cells from osteopetrotic subjects: contrasting phenotypes with TCIRG1, CLCN7, and attachment defects.

UNLABELLED: We studied osteoclastic differentiation from normal and osteopetrotic human CD14 cells in vitro. Defects in acid transport, organic matrix removal, and cell fusion with deficient attachment were found. Analysis of genotypes showed that TCIRG1 anomalies correlated with acid transport defects, but surprisingly, organic matrix removal failure correlated with CLCN7 defects; an attachment defect had normal TCIRG1 and CLCN7. INTRODUCTION: Osteopetrotic subjects usually have normal macrophage activity, and despite identification of genetic defects associated with osteopetrosis, the specific developmental and biochemical defects in most cases are unclear. Indeed, patients with identical genotypes often have different clinical courses. We classified defects in osteoclast differentiation in vitro using four osteopetrotic subjects without immune or platelet defects, three of them severe infantile cases, compared with normals. MATERIALS AND METHODS: Osteoclast differentiation used isolated CD14 cells; results were correlated with independent analysis of two key genes, CLCN7 and TCIRG1. CD14 cell attachment and cell surface markers and extent of differentiation in RANKL and colony-stimulating factor (CSF)-1 were studied using acid secretion, bone pitting, enzyme, and attachment proteins assays. RESULTS AND CONCLUSIONS: CD14 cells from all subjects had similar lysosomal and nonspecific esterase activity. With the exception of cells from one osteopetrotic subject, CD14 cells from osteopetrotic and control monocytes attached similarly to bone or tissue culture substrate. Cells from one osteopetrotic subject, with normal CLCN7 and TCIRG1, did not attach to bone, did not multinucleate, and formed no podosomes or actin rings in RANKL and CSF-1. Attachment defects are described in osteopetrosis, most commonly mild osteopetrosis with Glantzman's thrombasthenia. However, this case, with abnormal integrin alphavbeta3 aggregates and no osteoclasts, seems to be unique. Two subjects were compound heterozygotes for TCIRG1 defects; both had CD14 cells that attached to bone but did not acidify attachments; cell fusion and attachment occurred, however, in RANKL and CSF-1. This is consistent with TCIRG1, essential for H+-ATPase assembly at the ruffled border. A compound heterozygote for CLCN7 defects had CD14 cells that fused in vitro, attached to bone, and secreted acid, TRACP, and cathepsin K. However, lacunae were shallow and retained demineralized matrix. This suggests that CLCN7 may not limit H+-ATPase activity as hypothesized, but may be involved in control of organic matrix degradation or removal.

Autocrine and paracrine nitric oxide regulate attachment of human osteoclasts.

Nitric oxide (NO) can reduce bone loss in chronic bone diseases. NO inhibits or kills osteoclasts, but the mechanism of action of NO in human bone turnover is not clear. To address this, we studied effects of NO on attachment and motility of human osteoclasts on mineralized and tissue culture substrates under defined conditions. Osteoclasts were differentiated in vitro from CD14 selected monocytes in RANKL and CSF-1, and characterized by cathepsin K expression, tartrate-resistant acid phosphatase (TRAP) activity, acid secretion, and lacunar resorption. Cell attachment was labeled with monoclonal antibody 23C6, specific for a binding domain of a key osteoclast attachment protein, the CD51/CD61 integrin dimer (alpha(v)beta(3)), with or without cell permeabilization. A ring of integrin attachment during bone degradation delimits an extracellular acid compartment, while alpha(v)beta(3) forms focal attachments on non-resorbable substrates. On resorbable substrate but not non-resorbable substrate, alpha(v)beta(3) labeling required cell permeabilization, in keeping with the membrane-matrix apposition that excludes large molecules and allows extracellular acidification. Acid secretion was labeled with the fluorescent weak base indicator lysotracker. NO donors, S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP), downmodulated acid secretion simultaneously with cytoskeletal rearrangement, with alpha(v)beta(3) redistributed to a discontinuous pattern that labeled, on bone substrate, without membrane permeabilization. These effects were reversible, and an inhibitor of NO synthesis, N(G)-monomethyl-L-arginine (l-NMMA), increased acid secretion and decreased heterogeneity of attachment structures, showing that NO is an autocrine regulator of attachment. A hydrolysis-resistant activating cGMP analog 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate replicated effects of NO donors, while an inhibiting analog, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp-isomer, opposed them. On tissue culture or mineralized substrates, NO or cGMP analogs directly regulated motility; after washout cells reattached and survived for days. We conclude that NO is produced by human osteoclasts and regulates acid secretion and cellular motility, in keeping with autocrine and paracrine NO regulation of the resorption cycle.

Estrogen receptor-beta modulates synthesis of bone matrix proteins in human osteoblast-like MG63 cells.

Estrogens have complex effects on the skeleton, including regulation of modeling and maintenance of bone mass, which vary with cell type and developmental stage. Osteoblasts are key regulators of skeletal matrix synthesis and degradation. However, whether osteocytes, osteoblasts or earlier progenitors mediate estrogen effects, and the importance of estrogen receptors (ERs) alpha and beta, remain unclear. To address estrogen response in human cells closely related to secretory osteoblasts, we studied MG63 cells with ERalpha or ERbeta reduced to low levels by stable transfection of antisense plasmids. Collagen and alkaline phosphatase expression increased with estrogen in wild-type and ERalpha-suppressed cells, but not in ERbeta-suppressed cells. Matrix secretion occurs as osteoblasts cease dividing, and, in keeping with this, cell proliferation was reduced by estrogen except in ERbeta-antisense cells. No effects of estrogen on wild type or ER-suppressed cells were seen in expression of BMP 2, the BMP antagonist noggin, or Indian hedgehog, products that regulate differentiation of osteoblasts. In contrast to expectations that estrogen would modulate bone degradation, RANKL, CSF-1, and osteoprotegerin did not respond measurably to estrogen, regardless of ER status. In keeping with this result, estrogen response was not observed in assays of osteoclast development from CD14 cells supported by wild-type or ER-silenced MG63 cells. Since estrogens are major regulators of bone degradation in vivo, estrogen effects on osteoclasts may depend on interaction with stimuli present in bone but absent in the model studied. cDNA hybridization showed that additional estrogen-binding proteins including ERRalpha and BCAR3 were expressed by MG63, but estrogen effects in ERbeta-silenced cells were small, so these proteins are either minor regulators in MG63 cells, or act in concert with stimuli in addition to estrogen. We conclude that, in the MG63 cell line, estrogen increases synthesis of matrix proteins via ERbeta, and that, in the absence of additional stimuli, these cells are not major mediators of estrogen effects on osteoclast differentiation. Further, ERalpha is probably much more important in earlier stages of skeletal development, such as growth plate response, than in osteoblasts.